JAK-3 inhibitors for treating allergic disorders

ABSTRACT

Inhibitors of JAK3 kinase for the treatment of allergy inhibit mast cell degranulation an dmediator release.

FIELD OF THE INVENTION

This invention relates to compositions and methods for inhibiting JAK-3tyrosine kinase and to the treatment of allergic disorders byadministering an inhibitor of Janus Kinase 3 (JAK3).

BACKGROUND OF THE INVENTION

Signal transducers and activators of transcription (STAT) arepleiotropic transcription factors which mediate cytokine-stimulated geneexpression in multiple cell populations (D. A. Levy, Cytokine GrowthFactor Rev., 8:81 (1997)). All STAT proteins contain a DNA bindingdomain, a Src homology 2 (SH2) domain, and a transactivation domainnecessary for transcriptional activation of target gene expression.Janus kinases (JAK), including JAK1, JAK2, Tyk, and JAK3, arecytoplasmic protein tyrosine kinases (PTKs) which play pivotal roles ininitiation of cytokine-triggered signaling events by activating thecytoplasmic latent forms of STAT proteins via tyrosine phosphorylationon a specific tyrosine residue near the SH2 domain (See J. N. Ihle etal., Trends Genet., 11:69 (1995); J. E. Darnell et al., Science,265:1415 (1994); J. A. Johnston et al., Nature, 370:1513 (1994)).Tyrosine phosphorylated STAT proteins dimerize through specificreciprocal SH2-phosphotyrosine interactions and translocate from thecytoplasm to the nucleus where they stimulate the transcription ofspecific target genes by binding to response elements in their promoters(See W. J. Leonard, Nature Medicine, 2:968 (1996); Z. Zhong et al., PNASUSA, 91:4806 (1994) Darnell et al., Science, 264:1415 (1994)).

Among the four members of the JAK family, JAK3 is abundantly expressedin lymphoid cells and plays an important role in normal lymphocytedevelopment and function, as evidenced by qualitative and quantitativedeficiencies in the B-cell as well as T-cell compartments of the immunesystem of JAK3-deficient mice (T. Nosaka et al., Science, 270:800(1995); D. C. Thomas et al., Science, 270:794 (1995)) and development ofsevere combined immunodeficiency in JAK3-deficient patients (R. H.Buckley et al., J. Pediatr., 130:379 (1997)). Besides lymphoid cells,non-lymphoid cells, including monocytes, megakaryocytes, endothelialcells, cancers cells, and, as described herein, mast cells also expressJAK3, but no information is currently available regarding thephysiologic function of JAK3 in these non-lymphoid cell populations.See, D. C. Thomas et al., Curr. Opin. Immunol., 9:541 (1997); J. N.Hile, Philos. Trans. R. Soc. Lond B Biol. Sci., 351:159 (1996); J. W.Verbsky et al., J. Biol. Chem., 271:13976 (1996).

JAK-3 maps to human chromosome 19p12-13.1. A cluster of genes encodingprotooncogenes and transcription factors is also located near thisregion. JAK-3 expression has been demonstrated in mature B-cells as wellas B-cell precursors. JAK-3 has also been detected in leukemic B-cellprecursors and lymphoma B-cells. The physiological roles for JAK-3 havebeen borne out through targeted gene disruption studies in mice, thegenetic analysis of patients with severe combined immunodeficiency, andbiochemical studies of JAK-3 in cell lines. A wide range of stimuliresult in JAK-3 activation in B-cells, including interleukin 7 andinterleukin 4. The B-cell marker CD40 constitutively associates withJAK-3 and ligation of CD40 results in JAK-3 activation, which has beenshown to be mandatory for CD40-mediated gene expression. Constitutiveactivity of JAK-3 has been observed in v-abl transformed pre-B cells andcoimmunoprecipitations show that v-abl physically associates with JAK-3,implicating JAK-3 in v-abl induced cellular transformation. See J. N.Ihle, Philos Trans R Soc Lond B Biol Sci, 351:159 (1996); W. J. Leonardet al., Cytokine Growth Factor Rev., 8:81 (1997); M. C. Riedy et al.,Genomics, 37:57 (1996); M. G. Safford et al. [published erratum appearsin Exp. Hematol., 1997 July; 25(7):650] Exp. Hematol., 25:374 (1997); A.Kumar et al., Oncogene, 13:2009 (1996); S. M. Hoffman et al., Genomics,43:109 (1997); P. J. Tortolani et al., J. Immunol., 155:5220 (1995); N.Sharfe et al., Clin. Exp. Immunol., 108:552 (1997); C. B. Gurniak etal., Blood, 87:3151 (1996); C. Rolling et al., Oncogene, 10:1757 (1995);C. Rolling et al., FEBS Lett., 393:53 (1996); S. H. Hanissian et al.,Immunity, 6:379 (1997); N. N. Daniel et al., Science, 269:1875 (1995).

Acute allergic reactions, also known as immediate (type I)hypersensitivity reactions, including anaphylaxis with a potentiallyfatal outcome, are triggered by three major classes of proinflammatorymediators, namely preformed, granule-associated bioactive amines (e.g.,histamine, serotonin) and acid hydrolases (e.g., β-hexosaminidase),newly synthesized arachidonic acid metabolites [e.g., leukotriene (LT)C₄, prostaglandin D₂, and platelet activating factor], and a number ofproinflammatory vasoactive cytokines (e.g., tumor necrosis factor [TNF]α, interleukin-6 [IL-6]) (R. Malavija et al., J. Biol. Chem., 268:4939(1983); S. J. Galli et al., N. Eng J. Med., 328:257 (1993)). Theseproinflammatory mediators are released from sensitized mast cells uponactivation through the antigen-mediated crosslinking of their highaffinity cell surface IgE receptors/Fcε RI (M. J. Hamany et al.,Cellular Signaling, 7:1535 (1995); A. M. Scharenberg et al., Clin.Immunol., G. Marone ed., Basel, Karger (1995) at p. 72)). IgEreceptor/FcεRI is a multimeric receptor with α, β, and homodimeric γchains (See U. Blank et al., Nature, 337, 187 (1989)). Both β- and γsubunits of the IgE receptor/FcεRI contain ITAMs (ImmunoreceptorTyrosine-based Activation Motifs) which allow interaction with proteintyrosine kinases (PTK) and PTK substrates via their SH2 domains (See, N.Hirasawa et al., J. Biol. Chem., 270:10960 (1995)). The engagement ofIgE receptors by antigen triggers a cascade of biochemical signaltransduction events, including activation of multiple PTK (S. E.Lavens-Philips et al., Inflamm. Res., 47:137 (1998)). The activation ofPTK and subsequent tyrosine phosphorylation of their downstreamsubstrates have been implicated in the pathophysiology of type Ihypersensitivity reactions (See K. Moriya et al., PNAS USA, 94:12539(1997) Costello et al., Oncogene, 13:2595 (1996)).

Treatments for allergy are generally aimed at three possiblecomponents: 1) avoidance or reduced exposure to the allergen, which canbe very difficult especially in case of children; 2) allergenimmunotherapy, which only works for some allergens, and which isfrequently ineffective; and 3) pharmacotherapy (medication), which isthe most effective treatment. Allergy medication either should preventthe release of allergy causing chemicals such as histamine from mastcells, or stop the response of histamine on the tissues. Most currentlyavailable medications are anti-histamines. Usually in case of allergyand asthma, doctors prescribe second generation anti-histamines such asloraitidine (Claratin®), fexofenidine (Allegra®) or leukotrienesynthesis inhibitors such as zafirlukast (Accolate®) and zileutron(Zyflo®). Because the airway is more sensitive to leukotrienes producedby a number of inflammatory cells, anti-leukotriene agents are usuallymore effective for asthmatic conditions.

Although the second generation antihistamines are as effective as theolder ones (Benadryl and Chlortrimeton) they have two potentialproblems. First, these drugs only counteract the effect of histaminereleased by mast cells in the body, which are responsible for many butnot all the symptoms of allergy. Therefore, anti-histamines are veryeffective in decreasing the itching, sneezing, and nasal secretions, butdo not provide relief from nasal stuffiness and late phase allergicreactions. A number of inflammatory mediators other than histamine, suchas leukotrienes and a number of vasoactive cytokines, are also releasedby mast cells and basophils. These inflammatory mediators remainunaffected by anti-histamines and contribute significantly to thepatho-physiology of allergy and asthma. Sometimes a combination ofanti-allergic and inflammatory drugs works better, but at the same timethese combinations cause adverse side effects. Secondly, in more severeallergic reactions (anaphylaxis), anti-histamines do not havetherapeutic effect.

Until recently, therapy for asthma was based on the drug theophylline.This drug is an excellent, time proven, time tested drug but hadnumerous side effects. In the 1980's short acting beta-adrenergiccompounds were introduced. In the 1990's asthma therapy shifted, so thatasthmatic patients started taking cortocosteroids, cromolyn, andtheophylline in different combinations. Recently three anti-leukotrienedrugs: Accolate, Zyflo and Singulair, were introduced in to the U.S.market for the treatment of asthma. These medications either block therelease of leukotrienes (Zylec) or block their effect on tissues(Accolate). Each is available in tablet form to be taken 2 to 4 times aday. Although leukotriene inhibitors do not inhibit early phase ofallergy or asthmatic reaction, they are found to improve pulmonaryfunction and asthma symptoms and significantly reduce requirement ofbeta-agonist by reducing the bronchial hyper-responsiveness.

Despite the above described advances in therapy, there is currently aneed for therapeutic agents and methods that are useful for preventingor reducing immediate (type I) hypersensitivity reactions, includinganaphylaxis and other allergic reactions.

SUMMARY OF THE INVENTION

We have now discovered that IgE/antigen induced degranulation andmediator release are substantially reduced in Jak3^(-/-) mast cells fromJAK3-null mice that generated by targeted disruption of Jak3 gene inembryonic stem cells. Furthermore, treatment of mouse, rat, as well ashuman mast cells with4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline) (WHI-P131), arationally designed potent and specific inhibitor of JAK3, inhibiteddegranulation and proinflammatory mediator release after IgEreceptor/FcεRI crosslinking. In vivo administration of this potent JAK3inhibitor prevented mast cell degranulation and development ofcutaneous, as well as systemic, fatal anaphylaxis in mice. Thus, JAK3plays a pivotal role in IgE receptor/FcεRI mediated mast cell responsesboth in vitro and in vivo.

Treatment with JAK-3 inhibitor reduces and/or prevents allergicreactions and anaphylxis. JAK-3 inhibition results in reduced orinhibited degranulation and proinflamatory mediator release. Thus,targeting JAK-3 with a specific inhibitor provides a new and effectivetreatment and prevention for mast-cell mediated allergic reactions.

The invention provides a method comprising inhibiting mast cellactivation or degranulation by contacting the mast cell (in vitro or invivo) with an effective amount of a JAK-3 inhibitor.

The invention also provides a therapeutic method comprising treating apathology wherein mast cell activation or degranulation is implicatedand inhibition of mast cell activation or degranulation is desired byadministering a JAK-3 inhibitor to a mammal (e.g. a human) in need ofsuch therapy.

The invention also provides substances that are effective to inhibitJAK-3 for use in medical therapy, preferably for use in treatingconditions associated with mast cell activation or degranulation, suchas immediate hypersensitivity reactions. The invention also provides theuse of a substance that inhibits JAK-3 for the manufacture of amedicament for the treatment of a condition that is associated with mastcell activation or degranulation.

The invention also provides novel compounds of formula I, as disclosedhereinbelow, as well as processes and intermediates useful for theirpreparation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. FIG. 1A is a model of the JAK3 kinase domain showingmolecular surface of protein (blue) and catalytic (ATP binding) site(yellow).

FIG. 1B is a ribbon representation (Ca backbone) of the homology modelof the JAK3 kinase domain. The WHI-P131 molecule is shown as a spacefilling model in the catalytic site of JAK3.

FIG. 1C is a close-up view of the catalytic site of the JAK3 model witha docked inhibitor, WHI-P131 (green). Residues and inhibitor are shownas space filling atoms. The solvent-exposed opening of the catalyticsite has dimensions to allow a relatively planar inhibitor to enter andbind to JAK3. The opening of the pocket is defined by residues Pro906,Ser907, Gly908, Asp912, Arg953, Gly829, Leu828, and Tyr904 (blueresidues). The far wall deep inside the pocket is lined with Leu905(backbone portion), Glu903, Met902, Lys905, and Asp967 (pink residues),and the floor of the pocket is lined by Leu905 (side chain portion),Val884, Leu956, and Ala966 (yellow residues). Residues defining the roofof the pocket include Leu828, Gly829, Lys830, and Gly831 (uppermost blueresidues). Prepared using InsightII program.

FIGS. 2A-2C. FIG. 2A is a model of unoccupied space in the catalytic(ATP binding) site of a JAK3 homology model. Shown in green is thebinding site for ATP and the most likely binding site fordimethoxyquinazoline inhibitors. The green kinase active site regionrepresents a total volume of approximately 530 Å³. Modeling studiesshowed that an inhibitor or a portion of an inhibitor with significantbinding to this region would occupy a volume less than 530 Å³ and havemolecular dimensions compatible with the shape of the binding siteregion. Other regions near the binding site which show measurableunoccupied volume are shown in royal blue, pink, yellow, and light blue.These binding regions are either unavailable to inhibitor molecules(royal blue) or represent regions just large enough to occupy solventmolecules (pink, yellow, light blue). A model showing the inhibitorWHI-P131 docked into the catalytic site is shown in white, superimposedon the green region.

FIG. 2B is a model of the catalytic site of JAK3 with dockedquinazolines WHI-P131 (multicolor), WHI-P132 (pink), and WHI-P154(yellow). Each compound fits into the binding site, but WHI-P132 (shownto be inactive against JAK3 in biological assays). WHI-P132 lacks an OHgroup in a location to bind with Asp967. WHI-P131 and WHI-P154, with OHgroups at the C4' position of the phenyl ring, are able to form afavorable interaction with Asp967 of JAK3, which may contribute to theirenhanced inhibition activity.

FIG. 2C is a diagram showing features of dimethoxy quinazolinederivatives which are predicted to aid binding to JAK3 catalytic site.

FIGS. 3A-3B. FIG. 3A shows a structural comparison of nonconservedresidues in the catalytic sites of 5 different protein tyrosine kinases:JAK3 (pink), BTK (red), SYK (light blue), IRK (dark blue), and HCK(yellow). Residues within 5 Å of the docked JAK3 inhibitor, WHI-P131(white), are shown as rod-shaped side chains. The C alpha backbone ofJAK3 is shown as a thin pink line, for perspective. Regions A to Fcorrespond to areas containing nonconserved residues in the catalyticsite (see B and the Examples below). Crystal structure coordinates ofHCK (Sicheri et. al., 1997, Nature 385:602-609) and IRK (Hubbard et.al., 1997, Nature 372:746-754), and homology models of JAK3, BTK(Mahajan et. al., 1999, J. Biol. Chem., in press) and SYK were used forthe structural analysis.

FIG. 3B shows nonconserved residues in the catalytic sites of 8different protein tyrosine kinases. Regions A-F refer to locations inthe catalytic site which are illustrated in A.

FIGS. 4A-4B show expression and activation of JAK3 in mast cells afterIgE receptor crosslinking.

In FIG. 4A, RBL-2H3 mast cells were stained with a polyclonal anti-JAK3antibody and labeled with a fluorescein labeled secondary antibody aswell as the DNA specific dye toto-3 and visualized using confocal laserscanning microscopy.

In FIG. 4B, to study IgE/antigen induced activation of JAK3 in mastcells, RBL-2H3 mast cells were sensitized with monoclonal anti-DNP IgEand then challenged with DNP-BSA. Mast cells were lysed using aNonidet-P40 lysis buffer prior to or 30 minutes after antigen challenge,and JAK3 immune complexes from these cell lysates were subjected toanti-phosphotyrosine (APT) Western blot analysis to examine theautophosphorylation of the JAK3 kinase (Lanes 1 and 2). In parallel,JAK3 immune complexes were also examined by anti-JAK3 immunoblotting(Lanes 3 and 4) to confirm that the increased tyrosine phosphorylationin APT blots was not due to differences in the amount of JAK3immunoprecipitated.

FIGS. 5A-5D show IgE receptor/FcεRI-mediated responses of wild-type andJak3^(-/-) mast cells. Mast cells were cultured from the bone marrows ofJAK3-null (Jak3^(-/-)), and wild-type (Jak3^(+/+)) control mice. FIGS.5A1 and 5A2 show flow cytometric comparison of IgE receptor expressionon Jak3^(+/+) 5A1 versus Jak3^(-/-) 5A2 mast cells. Mast cell bound IgEwas stained with anti IgE-FITC antibody (PharMingen Laboratories). FIG.5B is a graph showing the proliferative responses of Jak3^(-/-) andJak3^(+/+) mast cells to SCF determined using the MTT assay system.FIGS. 5C and 5D are graphs showing activity of mast cells. Mast cellsfrom Jak3^(+/+) mice were sensitized with a monoclonal anti-DNP IgE, andthen challenged with DNP-BSA, as described in detail in the Examplesbelow. In parallel, Jak3^(-/-) mast cells were also sensitized andchallenged with antigen in the same fashion. Histamine (shown in 5C) andleukotriene (LT) C₄ (shown in 5D) levels were estimated in cell freesupernatants of BMMC. The values (mean±SEM; n=3) for spontaneoushistamine release from Jak3^(-/-) mast cells and control Jak3^(+/+) mastcells were 84.4±12.4 pg/μg protein and 19.5±1.8 pg/μg proteinrespectively. The IgE/antigen-induced degranulation of bone marrow mastcells was expressed as degranulation index and was calculated using theformula: Degranulation index=Histamine release after antigenchallenge/Spontaneous histamine release without antigen challenge. Mastcells from Jak3^(+/+) mice released 41.9±14.2 ng LTC₄ /10⁶ cells. TheLTC₄ release was expressed as percent of control.

FIGS. 6A-6E show the specificty of WHI-P131 of JAK3. In FIGS. 6A-6D,JAK3, JAK1, and JAK2 were immunoprecipitated from Sf21 insect ovarycells transfected with the appropriate baculovirus expression vectorsand treated with WHI-P131, then subjected to in vitro kinase assays asdescribed in the Examples below. The enzymatic activity of JAKs wasdetermined by measuring autophosphorylation in a 10 minute kinase assay,as described in the Examples below. The kinase activity (KA) levels wereexpressed as percentage of baseline activity (% CON). In FIG. 6E, EMSAsof 32Dc22-IL-2Rβ cells are shown. WHI-P131 (100 μg/ml) and WHI-P154 (100μg/ml) (but not WHI-P132; 100 μg/ml) inhibited IL-2 triggeredJAK-3-dependent STAT activation but not IL-3-triggeredJAK-1/JAK-2-dependent STAT activation in 32Dc11-IL-2Rβ cells.

FIG. 7 shows the specificity of WHI-P131 for JAK-3. JAK3, SYK, and BTKimmunoprecipitated from Sf21 insect ovary cells transfected with theappropriate baculovirus expression vectors, LYN immunoprecipitated fromNALM-6 human B-lineage ALL cells, and IRK immunoprecipitated from HepG2hepatoma cells were treated with WHI-P131, then subjected to in vitrokinase assays, as described in the Examples below.

FIGS. 8A-8D demonstrate that WHI-P131 prevents JAK3 but not SYKActivation in Mast cells after IgE Receptor crosslinking. RBL-2H3 cellswere sensitized with monoclonal anti-DNP IgE, treated with eithervehicle (8A) or 30 μM WHI-P131 (8B) and then challenged with DNP-BSA.Cells were lysed, and JAK3 immune complexes were subjected to kinaseassays in the presence of cold ATP followed by APT immunoblotting (upperpanels in 8A & 8B) as well as to JAK3 Western blot analysis (lowerpanels in 8A & 8B) as described in the Examples below. To show theactivation of SYK in mast cells after IgE receptor crosslinking, RBL-2H3cells were sensitized with monoclonal anti-DNP IgE, left untreated (8C)or treated with either vehicle or 30 μM WHI-P131 (8D) and thenchallenged with DNP-BSA. Cells were lysed at the indicated time points,and SYK immune complexes were subjected to kinase assays in the presenceof cold ATP followed by APT immunoblotting (upper panels in 8C & 8D) aswell as to SYK Western blot analysis (lower panels in 8C & 8D). C:Baseline control.

FIG. 9 shows the effect of the JAK3 inhibitor, WHI-P131, onIgE/antigen-induced activation of RBL-2H3 mast cells. RBL-2H3 cells werecultured overnight on 22×22 mm coverslips at a cell density of 0.01×10⁶/ml with 0.24 mg/ml DNP-IgE. Sensitized RBL-2H3 cells were then treatedwith 30 μM WHI-P131, vehicle, or control compounds WHI-P258 and WHI-P112prior to challenge with DNP-BSA, as described in the legend of FIG. 4.After stimulation with DNP-BSA for 1 hour, cells were fixed in coldmethanol for 15 minutes followed by permeabilization with PBS containing0.1% Triton X-100. Cells were incubated with a monoclonal antibodyrecognizing alpha-tubulin (clone B-5-1-2, Sigma Chemicals, St. Louis,Mo.) for 40 minutes at 37° C. After washing 3 times with PBS-0.1% TritonX-100, cells were incubated with fluorescein labeled secondary antibody(Zymed, San Francisco, Calif.) for another 40 minutes. Cells were washedthree times to remove unbound antibody. DNA labeling was performed byincubation of coverslips with toto-3 (Molecular Probes, Eugene, Oreg.)for 10 minutes. Excessive dye was washed with PBS-0.1% Triton X-100.Cells were visualized under MRC 1024 Laser Scanning Microscope aftermounting with Vectashield (Vector laboratories, Inc, Burlingame,Calif.), as previously reported.

FIG. 10 shows the effect of JAK3 inhibitor WHI-P131 on IgEreceptor/FcεRI-mediated calcium response in mast cells. IgE sensitizedRBL-2H3 cells were loaded with 1 μM Fluo-3 for 1 hour as described inthe Examples below. The cells were then challenged with DNP-BSA orionomycin and change in fluorescence was recorded. To study the effectof WHI-P131 on intracellular calcium mobilization, IgE-sensitized andFluo-3 loaded RBL-2H3 cells were incubated with 3 μM, 12 μM or 30 μMWHI-P131 for 5 minutes prior to challenge with DNP-BSA or ionomycin.

FIGS. 11A-11C show the effect of the JAK3 inhibitor WHI-P131 on IgEreceptor/FcεRI-mediated mast cell responses. RBL-2H3 cells weresensitized with monoclonal anti-DNP IgE, treated with WHI-P131, vehicleor control compounds, and then challenged with DNP-BSA, as described indetail in the Experimental Procedures section. FIG. 11A is a graphshowing mast cell degranulation (β-hexosaminidase release, % of total),was assessed by measuring the β-hexosaminidase levels in cell freesupernatants and Triton X-100 solubilized pellets using the formula:β-hexosaminidase release, % of total=100×(β-hexosaminidase level insupernatant/β-hexosaminidase level in supernatant+solubilized pellet).Vehicle treated control RBL-2H3 cells released 45.1±3.1% of theirhexosaminidase contents after DNP-BSA challenge. FIG. 11B shows LTC₄ andFIG. 11C shows TNF-α levels measured in cell-free supernatants. Vehicletreated control cells released 11.3±1.3 pg LTC₄, and 160±33.0 pgTNFα/10⁶ mast cells. The results on LTC₄ and TNFα release are expressedas percent of maximum control release from vehicle treated control mastcells. The data points represent the mean±SEM values obtained from 3-6independent experiments. *P<0.01 compared to control as determined byStudent's t test. *P<0.05 and **P<0.0001 compared to control asdetermined by Student's t test.

FIGS. 12A-12C show the effect of the JAK3 inhibitor WHI-P131 on IgEreceptor/FcεRI-mediated Human Mast Cell Responses. Fetal liver derivedhuman mast cells were cytocentrifuged and fixed with carnoy's fixative.

FIG. 12A is a photomicrograph showing mast cells staining (blue) fortryptase. Fetal liver derived mast cells were stimulated with variousconcentrations of 22E7 anti-(FcεRI antibody) for 15 minutes in tyrodebuffer. In some experiments IgE sensitized mast cells were stimulated bychallenging with anti-IgE. To study the effect of WHI-P131, mast cellswere incubated with indicated concentrations of WHI-P131 prior tostimulation.

In FIG. 12B, mast cell tryptase release (% of total) was assessed bymeasuring the tryptase levels in cell free supernatants and solubilizedcell pellets by ELISA. The results are expressed as percent tryptaserelease (B.1; a representative of 3 independent experiments) and percentinhibition of tryptase release (B.2; N=3). The mean spontaneous tryptasereleased was 8.3±3.9%. In FIG. 12C, LTC₄ levels were measured incell-free supernatants by ELISA (N=6). The results are expressed aspercent of control (N=6). Control stimulated cells released was 29.3±14ng LTC₄ /10⁶ cells. The data points represent the mean±SEM values.

FIGS. 13A and 13B are graphs showing pharmacokinetic features of theJAK3 inhibitor WHI-P131 in mice. Plasma concentration-time profiles ofWHI-P131 in mice following intravenous [13A] or intraperitoneal [13B]administration (12.5 mg/kg; 5 mice per group). The pharmacokineticparameters, including the central volume of distribution (Vc), estimatedmaximum plasma concentration (C_(max)), elimination half-life (t_(1/2)β), systemic exposure level/area under plasma concentration-time curve(AUC), and bioavailability (F), are shown in the insets.

FIGS. 14A-14E show the effects of the JAK3 inhibitor WHI-P131 onanaphylaxis in mice.

FIG. 14A is a graph showing the effects of WHI-P131 onanaphylaxis-associated vascular hyperpermeability, examined byevaluating the cutaneous extravasation of albumin-bound Evans blue dyein mice (n=12). The plasma exudation indices were determined for vehicletreated as well as WHI-P131 treated mice, as described in the Examples.To study the effect of WHI-P131 on anaphylaxis, IgE sensitized mice wereinjected with two consecutive doses of 10 or 25 mg/kg WHI-P131 at 90minutes before and 30 minutes before the antigen challenge,respectively. Mice were then challenged with 100 μg DNP-BSA in 2% Evansblue dye and the plasma exudation indices were determined. The datapoints represent the mean±SEM values. The data are expressed as plasmaexudation index (times increase in optical density over PBS treatedears. The mean OD (at 620 nm) of vehicle treated ears was 0.22±0.04before and 0.918±0.05 after the IgE/antigen challenge. *p<0.05 comparedto vehicle treated controls.

FIG. 14B is a photograph showing plasma extravasation during systemicanaphylaxis, evaluated as described in the Examples. FIG. 14B1 shows afoot pad of a control mouse after intravenous injection of Evans bluedye alone. FIG. 14B2 shows a foot pad of a vehicle treated mouse aftercoadministration of DNP-BSA and Evans blue. FIG. 14B3 shows a foot padof a WHI-P131 treated mouse after coadministration of DNP-BSA and Evansblue.

FIG. 14C is a photograph showing results of histopathologic evaluationof mast cell degranulation. Ears were removed 1 hour after the antigenchallenge from vehicle treated as well as WHI-P131 treated mice.Formalin-fixed thin sections (3-5 μm) of ears from were stained withAvidin-FITC.

FIG. 4D is a graph showing blood histamine levels of sensitized miceanalyzed after antigen challenge. Blood was collected by retro-orbitalbleeding and histamine levels were measured by ELISA. Histamine levelsare expressed as nM. The histamine levels in the blood of PBS treatedmice and IgE/antigen stimulated mice was 493±131 and 7527±2102 nMrespectively. The results are the mean±SEM values; n=3.

FIG. 14E is a graph showing results of a study of the effect of JAK3inhibitor WHI-P131 on fatal anaphylaxis in mice. BALB/c mice weresensitized with 100 mg/kg bovine serum albumin in 200 μl of the adjuvantaluminum hydroxide gel (Reheis Inc., Berkeley, N.J.), which favors theproduction of IgE in response to the presented antigen. Ten days later,mice were treated with two doses of WHI-P131 (45 mg/kg) or vehicle 30minutes apart and then challenged with an i.v. injection of the 10 mg/kgBSA. Cumulative proportions of mice surviving anaphylaxis-free are shownaccording to the time after the antigen challenge. Life-table analysisand statistical comparisons using the log-rank test were performed, aspreviously reported (Uckun et. al., 1998, Clin. Cancer Res., 4:901-912;Uckun et. al., 1995, Science, 267:886-891).

FIG. 15 is a diagram showing features of quinazoline derivatives whichaid binding to the JAK3 catalytic site.

DETAILED DESCRIPTION

As used herein, the term "inhibit" means to reduce by a measurableamount, or to prevent entirely.

The term to "treat" comprises inhibiting or blocking at least onesymptom that characterizes an immediate hypersensitivity reaction oracute allergic reaction, in a mammal threatened by, or afflicted with,said reaction.

JAK3, a member of the Janus family protein tyrosine kinases, was foundto be abundantly expressed in mast cells and its enzymatic activity isenhanced by IgE receptor/FcεRI crosslinking. IgE/antigen induceddegranulation and mediator release were substantially reduced withJak3^(-/-) mast cells from JAK3-null mice that were generated bytargeted disruption of Jak3 gene in embryonic stem cells.

JAK3-null mice did not develop an anaphylactic reaction to bovinealbumin whereas wild-type mice did. Treatment of mouse, rat, as well ashuman mast cells with4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline) (Compound 1), apotent and specific inhibitor of JAK3, inhibited degranulation andproinflammatory mediator release after IgE receptor/FcεRI crosslinking.Effective mast cell inhibitory plasma concentrations of Compound 1 wereachieved in vivo at non-toxic dose levels.

In vivo administration of this potent JAK3 inhibitor prevented mast celldegranulation and development of cutaneous as well as systemic fatalanaphylaxis in mice.

Thus, JAK3 plays a pivotal role in IgE receptor/FcεRI mediated mast cellresponses, both in vitro and in vivo. Therefore, targeting JAK3 withspecific inhibitors, such as those of formula I, provide a basis for newand effective treatment as well as prevention programs for mast cellmediated allergic reactions.

Methods of inhibiting JAK-3 can be carried out in vitro. Such in vitromethods are useful for studying the biological processes associated withcell response to allergens and for identifying new therapeutic agents.The methods of the invention can also be carried out in vivo. Suchmethods are useful for studying the biological processes associated withcell response to allergens, as well as for treating acute pathologicalconditions in mammals (e.g., humans) that result from exposure toallergens.

Allergic disorders associated with mast cell activation include Type Iimmediate hypersensitivity reactions such as allergic rhinitis (hayfever), allergic urticaria (hives), angioedema, allergic asthma andanaphylaxis, i.e., "anaphylatic shock." These disorders are treated orprevented by inhibition of JAK-3 activity, for example, byadministration of a JAK-3 inhibitor.

According to the invention, the JAK-3 inhibitors may be administeredprophylactically, i.e., prior to onset of acute allergic reaction, orthe JAK-3 inhibitors may be administered after onset of the reaction, orat both times.

In a preferred embodiment, the JAK-3 inhibitor is targeted to cells thatinduce an inflamatory response, such as mast cells, eosinophils, Tcells, and B cells. The compound is targeted by conjugation to atargeting moiety. Useful targeting moieties are ligands whichspecifically bind mast cell antigens or cell surface ligands, such asCD48 or the SCF receptor. Anti CD48 antibodies or SCF Ligand thus areexamples targeting moieties useful in a JAK3 inhibitor conjugate todeliver JAK3 to mast cells.

In addition, the antibodies B43 (binds B cells), TXU (binds T cells),GMSCF (binds receptors on eosinophils) or anti CD13 antibody (binds mastcells) are useful.

Compounds of the Invention

The JAK-3 inhibitors useful in the methods of the invention include allcompounds capable of inhibiting the activity of JAK-3, it being wellknown in the art how to measure a compound's ability to inhibit JAK-3,for example, using standard tests similar to the test described in thein Examples below.

JAK-3 inhibitors that are useful in the methods of the invention include##STR1## compounds of formula I: wherein

X is HN, R₁₁ N, S, O, CH₂, or R₁₁ CH;

R₁₁ is hydrogen, (C₁ -C₄)alkyl, or (C₁ -C₄)alkanoyl;

R₁ -R₈ are each independently hydrogen, hydroxy, mercapto, amino, nitro,(C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, (C₁ -C₄)alkylthio, or halo; wherein twoadjacent groups of R₁ -R₅ together with the phenyl ring to which theyare attached may optionally form a fused ring, for example, forming anaphthyl or a tetrahydronaphthyl ring; and further wherein the ringformed by the two adjacent groups of R₁ -R₅ may optionally besubstituted by 1, 2, 3, or 4 hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl, (C₁ -C₄)alkoxy, (C₁ -C₄)alkylthio, or halo; and provided thatat least one of R₂ -R₅ is OH.

R₉ and R₁₀ are each independently hydrogen, (C₁ -C₄)alkyl, (C₁-C₄)alkoxy, halo, or (C₁ -C₄)alkanoyl; or R₉ and R₁₀ together aremethylenedioxy; or a pharmaceutically acceptable salt thereof, andprovided that at least one of R₂ -R₅ is OH.

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkanoyl, etc., denote bothstraight and branched groups; but reference to an individual radicalsuch as "propyl" embraces only the straight chain radical, a branchedchain isomer such as "isopropyl" being specifically referred to. (C₁-C₄)Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, iso-butyl,and sec-butyl; (C₁ -C₄)alkoxy includes methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, and sec-butoxy; and (C₁ -C₄)alkanoylincludes acetyl, propanoyl and butanoyl.

A specific group of compounds are compounds of formula I wherein R₁ -R₅are each independently hydrogen, mercapto, amino, nitro, (C₁ -C₄)alkyl,(C₁ -C₄)alkoxy, (C₁ -C₄)alkylthio, halogen, or hydroxy, provided one ofR₂ -R₅ is OH.

Another specific group of compounds are compounds of formula I whereinR₉ and R₁₀ are each independently hydrogen, (C₁ -C₄)alkyl, halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; or apharmaceutically acceptable salt thereof.

Preferred JAK-3 inhibitors include4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (P131),4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (P154),4-(3'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline, (P180) and4-(3',5'-dibromo-4'-hydroxy phenyl)-6,7-dinethoxyquinazoline (P97) or apharmaceutically acceptable salt thereof.

Second Generation of JAK3 Inhibitors

Using the model methods modeling JAK-3 binding described hereinbelow,one skilled in the art can prepare a number of prefered JAK-3 inhibitorsthat fit the JAK-3 binding pocket are thereby predicted to have potentinhibitory activity.

Compounds are designed to fit and fill the binding pocket and to provideareas for interaction with contact residues as shown in FIG. 15. Secondgeneration compounds of the invention, which were designed to fit andtarget the JAK-3 kinase active site, are predicted to interact favorablywith JAK-3 kinase residues. These compounds of the invention are showndiagramatically below and particularly in Table 2. The synthetic schemesfor producing these compounds are depicted below in schemes 3A-3D.

                  TABLE 2                                                         ______________________________________                                        Second generation quinazoline designs targeting JAK3 kinase active            site. All compounds were predicted to interact favorably with                 JAK3 kinase residues. NA = not applicable.                                    ______________________________________                                         ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                     Com-                                 Molecular                                                                            Molecular                         pound                                Surface                                                                              Volume                            Name  R3'   R4'    R5'   R6'   R7'   Area (Å.sup.2)                                                                   (Å.sup.3)                     ______________________________________                                        Q1    H     OH     OH    H     NA    275    273                               Q2    Br    H      OH    CH.sub.2 OH                                                                         NA    300    288                               Q3    Br    H      OH    NH.sub.2                                                                            NA    283    274                               Q4    Br    H      OH    NO.sub.2                                                                            NA    300    294                               Q5    H     OH     Br    OH    NA    322    308                               Q6    H     OH     Br    CH.sub.2 OH                                                                         NA    327    323                               Q7    H     OH     Br    NH.sub.2                                                                            NA    318    311                               Q8    H     OH     Br    NO.sub.2                                                                            NA    340    329                               Q9    H     OH     Br    H     NA    317    295                               Q10   H     OH     OH    H     NA    308    306                               Q11   H     OH     CH.sub.2 OH                                                                         H     NA    314    321                               Q12   H     OH     NH.sub.2                                                                            H     NA    309    309                               Q13   H     OH     NO.sub.2                                                                            H     NA    329    331                               Q14   H     OH     Br    H     OH    336    317                               Q15   H     OH     Br    H     CH.sub.2 OH                                                                         349    334                               Q16   H     QH     Br    H     NH.sub.2                                                                            336    321                               Q17   H     OH     Br    H     NO.sub.2                                                                            359    340                               ______________________________________                                         ##STR6##     Preferred substitutions are those where: R3'=H, Br, OH

R4'=H, OH, H, Br

R5'=OH

R6'=H, CH₂ OH, NH₂, NO₂

n¹ =1-3,

provided one of R₃, R₄, R₅ is OH. Preferably, there are 1-3substitutions on the phenyl ring. ##STR7## Preferred are: R5'=Br

R6'=OH, CH₂ OH, NH₂, NO₂

n¹ =1-3 ##STR8##

Preferably, R5'=Br, OH, CH₂ OH, NH₂, or NO₂. ##STR9## Preferably: R5'=Br

R7'=OH, CH₂ OH, NH₂, or NO₂

n¹ =1-3

Suitable JAK-3 inhibitors also include antibodies to JAK-3, andantisense oligonucleotides that inhibit JAK-3 expression and synthesis.

Compounds that inhibit JAK-3 can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, JAK-3 inhibitors may be systemically administered, e.g., orally,in combination with a pharmaceutically acceptable vehicle such as aninert diluent or an assimilable edible carrier, or by inhalation orinsufflation. They may be enclosed in hard or soft shell gelatincapsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the JAK-3 inhibitors may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.The JAK-3 inhibitors may be combined with a fine inert powdered carrierand inhaled by the subject or insufflated. Such compositions andpreparations should contain at least 0.1% JAK-3 inhibitor. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of JAK-3 inhibitor insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the JAK-3 inhibitor maybe incorporated into sustained-release preparations and devices.

The JAK-3 inhibitor may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the JAK-3inhibitor can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the JAK-3 inhibitor which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform must be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the JAK-3inhibitor in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the JAK-3 inhibitor may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the JAK-3 inhibitor can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the JAK-3 inhibitors to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the JAK-3 inhibitor in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the JAK-3 inhibitor required for use in treatment willvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The JAK-3 inhibitor is conveniently administered in unit dosage form;for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the JAK-3 inhibitor should be administered to achieve peakplasma concentrations of from about 0.5 to about 75 μM, preferably,about 1 to 50 μM, most preferably, about 2 to about 30 μM. This may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of the JAK-3 inhibitor, optionally in saline, or orallyadministered as a bolus containing about 1-100 mg of the JAK-3inhibitor. Desirable blood levels may be maintained by continuousinfusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the JAK-3 inhibitor.

The JAK-3 inhibitor may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Construction of a Homology Model for the JAK-3 KinaseDomain

Because the three dimensional coordinates of the JAK3 kinase domain arecurrently unknown, a structural model of JAK3 was required for a dockinganalysis of JAK3 inhibitors. A homology model of JAK3 was constructed byusing known coordinates of homologous kinase domains as a reference.

The design of the JAK3 homology model was carried out by first obtainingthe protein sequence of JAK3 (Swiss-Prot #P52333, Univ. of Geneva,Geneva, Switzerland) from GenBank (National Center for BiotechnologyInformation, Bethesda, Md.) and determining the most reasonable sequencealignment for the JAK3 kinase domain relative to some templatecoordinates [known kinase structures, HCK (Sicheri et. al., 1997, Nature385, 602-609), FGFR (Mohammadi et. al., 1996; Mohammadi et. al., 1997),and IRK (Hubbard, 1997, EMBO J. 16, 5572-5581)]. This was accomplishedby first superimposing the Cα coordinates of the kinase domains of HCK,FGFR, and IRK using the InsightII program (Molecular Simulation Inc,1996, San Diego, Calif.) to provide the best overall structuralcomparison. The sequences were then aligned based on the superimpositionof their structures (amino acid sequences were aligned together if theirCα positions were spatially related to each other). The alignmentaccommodated such features as loops in a protein which differed from theother protein sequences. The structural superimposition was performedusing the Homology module of the InsightII program and a SiliconGraphics INDIGO2 computer (Silicon Graphics, Mountain View, Calif.).

The sequence alignment was done manually and produced a sequencevariation profile for each superimposed Cα position. The sequencevariation profile served as a basis for the subsequent sequencealignment of the JAK3 kinase with the other three proteins. In thisprocedure, the sequence of JAK3 was incorporated into the program andaligned with the three known kinase proteins based on the sequencevariation profiles described previously.

Next, a set of 3D coordinates was assigned to the JAK3 kinase sequenceusing the 3D coordinates of HCK as a template and the Homology modulewithin the InsightII program. The coordinates for a loop region where asequence insertion occurs (relative to HCK without the loop) were chosenfrom a limited number of possibilities automatically generated by thecomputer program and manually adjusted to a more ideal geometry usingthe program CHAIN (Sack, 1988).

Finally, the constructed model of the JAK3 kinase domain was subjectedto energy minimization using the X-PLOR program (Brunger, 1992) so thatany steric strain introduced during the model-building process could berelieved. The model was screened for unfavorable steric contacts and ifnecessary such side chains were remodeled either by using a rotamerlibrary database or by manually rotating the respective side chains. Theprocedure for homology model construction was repeated for JAK1(SWISS-PROT #P23458) and JAK2 (Genbank #AF005216) using the JAK3 modelas a structural template. The energy minimized homology models of JAK1,JAK2, and JAK3 were then used, in conjunction with energy-minimizedstructural models of dimethoxyquinazoline compounds, for modelingstudies of JAK/dimethoxyquinazoline complexes.

FIGS. 1A & 1B show the JAK3 homology model of the kinase domain, whichis composed of an N-terminal lobe and a C-terminal lobe that are linkedby a hinge region near the catalytic (ATP-binding) site. The catalyticsite is a pocket located in the central region of the kinase domain,which is defined by two β-sheets at the interface between the N and Clobes. The opening to the catalytic site is solvent accessible andfacilitates binding of ATP. Small molecule inhibitors could also bind tothe catalytic site which would result in an attenuation of PTK activityby inhibiting the ATP binding.

An analysis of the JAK3 model revealed specific features of thecatalytic site which can be described as a quadrilateral-shaped pocket(FIG. 1C). The opening of the pocket is defined by residues Pro906,Ser907, Gly908, Asp9l2, Arg953, Gly829, Leu828, and Tyr904 (blueresidues in FIG. 1C). The far wall deep inside the pocket is lined withLeu905 (backbone portion), Glu903, Met902, Lys905, and Asp967 (pinkresidues, FIG. 1C). The floor of the pocket is lined by Leu905 (sidechain portion), Val884, Leu956, and Ala966 (yellow residues, FIG. 1C).The residues defining the roof of the pocket include Leu828, Gly829,Lys830, and Gly831 (uppermost blue residues, FIG. 1C). FIGS. 1C and 2Aillustrate that the catalytic site of the JAK3 model has approximatedimensions of 8 Å×11 Å×20 Å and an available volume for binding ofapproximately 533 Å³. According to the model, the solvent exposedopening to the binding region would allow inhibitors to enter and bindif the molecule contains some planarity. Asp 1017 can form a hydrogenbond with a 3' or 4' OH group of a quinazoline bound to the catalyticsite, and Leu955 can interact with a quinazoline ring nitrogenhydrophilic substituent at C5' and C6' on the phenyl ring of quinazolinewould also enhance binding to JAK3. Steric hinderence with Met 952prevents the addition of a non-hydrogen substituent at C2. Modificationsto increase the volume of the inhibitor in the pocket, for example to avolume of about 200 Å³ -500 Å³, 225-350 Å³ will provide more potentcompounds.

While most of the catalytic site residues of the JAK3 kinase domain wereconserved relative to other PTK, a few specific variations were observed(FIG. 3). These differences include an alanine residue in BTK, IRK, andHCK/LYN (region A, FIG. 3A) which changes to Glu in SYK and Pro906 inJAK3. At region B, a tyrosine residue is conserved in JAK3 (Tyr904),BTK, and LYN, but changes to Phe in HCK (which is the only apparentresidue difference between HCK and LYN which is relevant to inhibitorbinding), Met in SYK, and Leu in IRK. Region C shows a methionineresidue which is conserved in BTK, IRK, and HCK/LYN, but changes toLeu905 in JAK3 and to Ala in SYK. Region D shows Met902 in JAK3, whichis conserved in SYK and IRK but changes to Thr in BTK and to a muchsmaller residue, Ala, in LYN and HCK. This Met902 residue in JAK3, whichis located on the back wall of the pocket and protrudes in toward thecenter of the pocket volume, can significantly affect the shape of thebinding pocket. At this location, the extended conformation of theMet902 side chain can hinder the close contact of inhibitors withresidues lining the back wall of the pocket and the hinge region,relative to other kinases with smaller residues here such as BTK (Thr)and HCK/LYN (Ala).

Ala966 in region E is conserved in HCK/LYN but changes to Gly in IRK andto the more hydrophilic residue Ser in BTK and SYK. Region F, which isfarther away from the inhibitor location, is the least conserved regionof the catalytic site and contains Asp912 in JAK3, Asn in BTK, Lys inSYK, Ser in IRK, and Asp in HCK/LYN (FIG. 3). These residue identitydifferences among tyrosine kinases provide the basis for designingselective inhibitors of the JAK3 kinase domain.

JAK3 contains Ala 1016 in the binding site, which changes to glycine inJAK1 and JAK2. The slightly larger alanine residue of JAK3 can providelarger alanine residue of JAK3 can provide larger surface contact andhydrophobic interactions with inhibitors.

Example 2 Docking Procedure Using Homology Model of JAK3 Kinase Domain

Modeling of the JAK3/dimethoxyquinazoline complexes was done using theDocking module within the program INSIGHTII and using the Affinity suiteof programs for automatically docking an inhibitor into a kinase domainbinding site. An energy-minimized model for each inhibitor compound wasdocked into the homology model coordinates of the catalytic site ofJAK3. Each compound was docked into the active site of the JAK3 kinasedomain based on the position of quercetin in the HCK/quercetin crystalstructure (Sicheri et al., 1997, Nature, 385:602-609).

The hydrogens on JAK3 were generated and potentials were assigned toJAK3 and its inhibitor model prior to the start of the dockingprocedure. The docking method in the InsightII program uses the CVFFforce field and a Monte Carlo search strategy to search for and evaluatedocked structures. While the coordinates for the bulk of the receptorwere kept fixed, coordinates of a defined region of the binding sitewere allowed to adjust as simulated interactions with the inhibitor wereestimated. This binding region was defined as a zone 4.5 Å away from theinhibitor and allowed residues within this zone to shift and/or rotateto energetically favorable positions to accommodate the inhibitor.

After an assembly was defined which included the protein and itsinhibitor molecule, docking calculations were performed using the fixeddocking mode. Calculations which approximated hydrophobic and hydrogenbonding interactions were used to identify the five best dockedpositions for each compound in the JAK3 catalytic site. The variousdocked positions of each compound were evaluated using a Ludi (Bohm,1994) scoring procedure in INSIGHTII which estimates a binding constant,K_(i), taking into account lipophilic, hydrogen bonding, and van derWaals interactions between the inhibitor and the protein. A comparisonof the catalytic site residues of several different PTK was made bymanually superimposing crystal structure coordinates of the kinasedomains of HCK, and IRK (Hubbard, 1997, EMBOJ. 16:5572-81) and models ofJAK1, JAK2, JAK3, BTK (Mahajan, et. al., 1999, Mol. Cell. Biol. 15,5304-5311), and SYK (Mao, unpublished data), and then identifyingfeatures in the active site which were unique to JAK3 (FIG. 3).

The computer docking procedure was used to predict how well potentialinhibitors could fit into and bind to the catalytic site of JAK3 andresult in kinase inhibition (FIG. 2B). The dimethoxyquinazoline compoundWHI-P258 (4-(phenyl)-amino-6,7-dimethoxyquinazoline) contains twomethoxy groups on the quinazoline moiety but no other ring substituents.Molecular modeling studies using the homology model of JAK3 kinasedomain suggested that WHI-P258 would fit into the catalytic site ofJAK3, but probably would not bind very tightly due to limited hydrogenbonding interactions. Asp967, a key residue in the catalytic site ofJAK3, can form a hydrogen bond with molecules binding to the catalyticsite, if such molecules contain a hydrogen bond donor group such as anOH group. WHI-P258, however, does not contain an OH group and thereforewould not interact as favorably with Asp967. We postulated that thepresence of an OH group at the 4' position of the phenyl ring ofWHI-P258 would result in stronger binding to JAK3 because of addedinteractions with Asp967. A series of dimethoxyquinazoline compoundswere designed and synthesized to test this hypothesis.

An estimation of the molecular volume for the compounds is provided inTable 1. A summary of structural features of the designeddimethoxyquinazoline compounds which were observed to be relevant forbinding to the catalytic site of JAK3 is shown in FIG. 2C. Theapproximate molecular volumes of the compounds in Table 1 range from 252Å³ to 307 Å³, which are small enough to fit into the 530 Å³ binding siteof JAK3 kinase. Table 1 also lists the results of molecular modelingstudies including estimated binding constants (i.e., K_(i) values) forthe compounds which were docked into the JAK3 catalytic site. Thecompounds which were evaluated in docking studies contain substitutionsof similar functional groups at different positions on the phenyl ring.

                                      TABLE 1                                     __________________________________________________________________________    Predicted interaction of quinazolines with JAK3 kinase active site            and measured inhibition values (IC.sub.50 values) for JAK3 kinase.             ##STR10##                                                                                     Predicted                                                                          Molecular                                               Compound         binding to                                                                         Surface                                                                            Molecular                                          Name  R.sub.1                                                                         R.sub.2                                                                          R.sub.3                                                                          R.sub.4                                                                          JAK3 Area (Å.sup.2)                                                                 Volume (Å.sup.3)                                                                IC.sub.50 (μM)                            __________________________________________________________________________    VHI-P131                                                                            H OH H  H  favorable                                                                          726  261   78                                           VHI-P154                                                                            H OH Br H  favorable                                                                          296  284   128                                          VHI-P180                                                                            H H  OH H  favorable                                                                          273  260    3                                           VHI-P97                                                                             Br                                                                              OH Br H  favorable                                                                          314  307   11                                           VHI-P79                                                                             H H  Br H  less 278  272  >300                                                           favorable                                                    VHI-P111                                                                            H CH.sub.3                                                                         Br H  less 309  291  >300                                                           favorable                                                    VHI-P112                                                                            Br                                                                              H  H  Br less 306  297  >200                                                           favorable                                                    VHI-P132                                                                            H H  H  OH less 269  262  >300                                                           favorable 264                                                VHI-P258                                                                            H H  H  H  less 266  252  >300                                                           favorable                                                    __________________________________________________________________________

The conformations of the energy-minimized models of the compounds listedin Table 1 were relatively planar, with dihedral angles of approximately4-18° between the phenyl ring and quinazoline ring system. Thisconformation allows the compounds to fit more easily into the catalyticsite of JAK3. All of the listed compounds contain a ring nitrogen (N1),which can form a hydrogen bond with NH of Leu905 in the hinge region ofJAK3. When N1 is protonated, the NH can instead interact with thecarbonyl group in Leu905 of JAK3. The presence of an OH group at the 4'position on the phenyl ring was anticipated to be particularly importantfor binding to the catalytic site of JAK3. WHI-P131 (estimated K_(i)=2.3 μM), WHI-P154 (estimated K_(i) =1.4 μM), WHI-P97 (estimated K_(i)=0.6 μM) shown in Table 1 were predicted to have favorable binding toJAK3 and potent JAK3 inhibitory activity because they contain a 4' OHgroup on the phenyl ring which can form a hydrogen bond with Asp967 ofJAK3, contributing to enhanced binding. By comparison, the 2' OH groupof WHI-P132 is not in the right orientation to interact with Asp967 andit probably would form an intramolecular hydrogen bond with thequinazoline ring nitrogen, which may contribute to a significantly loweraffinity of WHI-P132 for the catalytic site of JAK3.

The relatively large bromine substituents (WHI-P97, WHI-P154) canincrease the molecular surface area in contact with binding siteresidues if the molecule can fit into the binding site. Modeling ofWHI-P154 and WHI-P97 showed that there is enough room to accommodate thebromine groups if the phenyl ring is tilted slightly relative to thefused ring group of the molecule.

Example 3 Chemical Synthesis and Characterization of JAK-3 Inhibitors

The results from the modeling studies prompted the hypothesis thatWHI-P131, WHI-P154, and WHI-P97 would exhibit potent JAK3-inhibitoryactivity. In order to test this hypothesis and validate the predictivevalue of the described JAK3 homology model, we synthesized WHI-P131,WHI-P154, WHI-P97, and 5 other dimethoxyquinazoline compounds listed inTable 1.

Chemical Synthesis of Quinazoline Derivatives

The common starting material, 4-chloro-6,7-dimethoxyquinazoline (1) forthe synthesis of all the WHI compounds, was prepared using publishedprocedures as shown in Scheme 1. ##STR11##

4,5-Dimethoxy-2-nitrobenzoic acid (3) was treated with thionyl chlorideand then reacted with ammonia to give 4,5-dimethoxy-2-nitrobenzamide (4)as described by F. Nomoto et al. Chem. Pharm. Bull., 1990, 38,1591-1595. The nitro group in compound (4) was reduced with sodiumborohydride in the presence of copper sulfate (see C. L. Thomas,Catalytic Processes and Proven Catalysts, Academic Press, New York(1970)) to give 4,5-dimethoxy-2-aminobenzamide (5) which was cyclizcd byrefluxing with formic acid to give 6,7-dimethoxyquinazoline-4(3H)-one(6). Compound (6) was refluxed with phosphorus oxytrichloride to providethe common synthetic precursor (7).

The compounds listed in Table 1 were all synthesized from4-chloro-6,7-dimethoxyquinazoline 1 according to Scheme 2, as previouslyreported (Narla et. al., 1998). In this procedure, a mixture of4-chloro-6,7-dimethoxyquinazoline 1 (448 mg, 2 mmols) and thesubstituted aniline (2.5 mmols) in EtOH (20 mL) was heated to reflux.Heating was continued for 4-24 hours. After cooling to room temperature,excess amount of Et₃ N was added to neutralize the solution and thesolvent was concentrated to give the crude product, which wasrecrystallized from DMF. ##STR12## Analytical Data for SynthesizedCompounds

Melting points are uncorrected. ¹ H NMR spectra were recorded using aVarian Mercury 300 spectrometer in DMSO-d₆ or CDCl₃. Chemical shifts arereported in parts per million (ppm) with tetramethylsilane (TMS) as aninternal standard at zero ppm. Coupling constants (J) are given in hertzand the abbreviations s, d, t, q, and m refer to singlet, doublet,triplet, quartet and multiplet, respectively. Infrared spectra wererecorded on a Nicolet PROTEGE 460-IR spectrometer. Mass spectroscopydata were recorded on a FINNIGAN MAT 95, VG 7070E-HF G.C. system with anHP 5973 Mass Selection Detector. UV spectra were recorded on BECKMAN DU7400 and using MeOH as the solvent. TLC was performed on a precoatedsilica gel plate (Silica Gel KGF; Whitman Inc). Silica gel (200-400mesh, Whitman Inc.) was used for all column chromatography separations.All chemicals were reagent grade and were purchased from AldrichChemical Company (Milwaukee, Wis.) or Sigma Chemical Company (St. Louis,Mo.).

4-Chloro-6,7-dimethoxyquinazoline 1

Yield 75.00%; mp 259.0-263.0° C.; ¹ H NMR (DMSO-d₆) d 8.75 (s, 1H, 2-H),7.53 (s, 1H, 5-H), 7.25 (s, 1H, 8H), 3.91 (s, 3H, --OCH₃), 3.89 (s, 3H,--OCH₃); IR (KBr) 2963, 2834, 1880, 1612, 1555, 1503, 1339, 1153, 962cm⁻¹ ; GC/MS m/z 224 (M⁺, 100), 209 (M⁺ --CH₃, 9), 189 (19), 169 (11);Anal. (C₁₀ H₉ ClN₂ O₂) C, H, N.

4,5-Dimethoxy-2-nitrobenzamide 3

Yield 88.50%; mp 197.0-200.0° C.; ¹ H NMR (DMSO-d₆) d 7.60 (s, 2H,-NH₂), 7.57 (s, 1H, 6-H), 7.12 (s, 1H, 3-H), 3.90 (s, 3H, --OCH₃), 3.87(s, 3H, --OCH₃); IR (KBr) 3454, 2840, 1670, 1512, 1274, 1227 cm⁻¹ ;GC/MS m/z 226 (M⁺, 10), 178(99), 163(100), 135(51).

6,7-Dimethoxyquinazoline-4(3H)-one 5

Yield 81.50%; mp 295.0-297.0° C.; ¹ H NMR (DMSO-d₆) d 12.03 (br, s, 1H,--NH), 7.99 (s, 1H, 2-H), 7.42 (s, 1H, 5-H), 7.11 (s, 1H, 8-H), 3.88 (s,3H, --OCH₃), 3.85 (s, 3H, --OCH₃); IR (KBr) 3015, 2840, 1648, 1504,1261, 1070 cm⁻¹ ; GC/MS m/z 206 (M⁺, 100), 191 (M⁺ --CH₃, 31), 163 (17),120 (15).

4-(3'-Bromophenyl)-amino-6,7-dimethoxyquinazoline WHI-P79

Yield 84.17%; mp 246.0-249.0° C.; ¹ H NMR (DMSO-d₆) d 10.42 (br, s, 1H,NH), 8.68 (s, 1H, 2-H), 8.07-7.36 (m, 5H, 5, 2',4',5',6'-H), 7.24 (s,1H, 8-H), 3.98 (s, 3H, --OCH₃), 3.73 (s, 3H, --OCH₃); IR (KBr) 3409,2836, 1632, 1512, 1443, 1243, 1068 cm⁻¹ ; GC/MS m/z 361 (M⁺ +1, 62),360(M⁺, 100), 359 (M⁺ -1, 64), 344(11), 222(11), 140(14); Anal. (C₁₆ H₁₄BrN₃ O₂) C, H, N.

4-(3',5'-Dibromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazolineWHI-P97

Yield 72.80%; mp>300.0° C.; ¹ H NMR (DMSO-d₆) d 9.71 (s, 1H, --NH), 9.39(s, 1H, --OH), 8.48 (s, 1H, 2-H), 8.07 (s, 2H, 2', 6'-H), 7.76 (s, 1H,5-H), 7.17 (s, 1H, 8-H), 3.94 (s, 3H, --OHC₃), 3.91 (s, 3H, --OCH₃); IR(KBr) 3504, 3419, 2868, 1627, 1512, 1425, 1250, 1155 cm⁻¹ ; GC/MS m/z456 (M⁺ +1, 54), 455 (M⁺, 100), 454 (M⁺ -1, 78), 439 (M⁺ --OH, 8), 376(M⁺ +1 --Br, 10), 375 (M⁺ --Br, 11), 360 (5); Anal. (C₁₆ H₁₃ Br₂ N₃ O₃)C, H, N.

4-(3'-Bromo-4'-methyphenyl)-amino-6,7-dimethoxyquinazoline WHI-P1111

Yield 82.22%; mp 225.0-228° C.; ¹ H NMR(DMSO-d₆) d 10.23 (s, 1H, --NH),8.62 (s, H, 2-H), 8.06 (d, 1H, J_(2'),6' =2.1 Hz, 2'-H), 7.89 (s, 1H,5-H), 7.71 (dd, 1H, J_(5'),6' =8.7 Hz, J_(2'),6' =2.1 Hz, 6'-H), 7.37(d, 1H, J_(5'),6' =8.7 Hz, 5'-H), 7.21 (s, 1H, 8-H), 3.96 (s, 3H,--OCH₃), 3.93 (s, 3H, --OCH₃), 2.33 (s, 3H, --CH₃); IR (KBr) 3431, 3248,2835, 1633, 1517, 1441, 1281, 1155 cm⁻¹ ; GC/MS m/z 375 (M⁺ +1, 77), 374(M⁺, 100), 373 (M⁺ -1, 77), 358 (M⁺ +1 --OH, 11), 357 (1), 356 (6);Anal. (C₁₇ H₁₆ BrN₃ O₂.HCl) C, H, N.

4-(2',5'-Dibromophenyl)-amino-6,7-dimethoxyquinazoline WHI-P112

Yield 70.05%; mp>300.0° C.; ¹ H NMR (DMSO-d6) d 11.51 (s, 1H, --NH),8.76 (s, 1H, 2-H), 8.21 (s, 1H, 5-H), 7.81 (d, 1H, J_(4'),6' =2.4 Hz,6'-H), 7.75(d, 1H, J_(3'), 4' =8.7 Hz, 3'-H), 7.55 (dd, 1H, J_(4'),6'=2.4 Hz, J_(3'),4' =8.7 Hz, 4'-H), 7.33 (s, 1H, 8-H), 3.98 (s, 3H,--OCH₃), 3.97 (s, 3H, --OCH₃); IR (KBr) 3444, 2836, 1628, 1510, 1431,1277, 1070 cm-1; GC/MS mz 440 (M⁺ +1, 10), 439 (M+, 7),438 (M⁺ -1, 4),360 (M⁺ +1 --Br, 99), 359 (M⁺ --Br, 20), 358 (M⁺ -1 --Br, 100), 343(21),299 (9); Anal. (C₁₆ H₁₃ Br₂ N₃ O₂.HCl) C, H, N.

4-(4'-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P131

Yield 84.29%; mp 245.0-248.0° C.; ¹ H NMR (DMSO-d₆) d 11.21 (s, 1H,--NH), 9.70 (s, 1H, --OH), 8.74 (s, 1H, 2-H), 8.22 (s, 1H, 5-H), 7.40(d, 2H, J=8.9 Hz, 2',6'-H), 7.29 (s, 1H, 8-H), 6.85 (d, 2H, J=8.9 Hz,3',5'-H), 3.98 (s, 3H, --OCH₃), 3.97 (s, 3H, --OCH₃); IR (KBr) 3428,2836, 1635, 1516, 1443, 1234 cm⁻¹ ; GC/MS m/z 298 (M⁺ +1, 100), 297(M⁺,27), 296(M⁺ -1, 12); Anal. (C₁₆ H₁₅ N₃ O₃.HCl) C, H, N.

4-(2'-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P132

Yield 82.49%; mp 255.0-258.0° C. ¹ H NMR (DMSO-d₆) d 9.78 (s, 1H, --NH),9.29 (s, 1H, --OH), 8.33 (s, 1H, 2-H), 7.85 (s, 1H, 5-H), 7.41-6.83 (m,4H, 3',4',5',6'-H), 7.16 (s, 1H, 8-H), 3.93 (s, 3H, --OCH₃), 3.92 (s,3H, --OCH₃); IR (KBr) 3500, 3425, 2833, 1625, 1512, 1456, 1251, 1068cm³¹ 1 ; GC/MS m/z 298 (M⁺ +1, 9), 297 (M⁺, 57), 281 (M⁺ +1 --OH, 23),280 (M⁺ --OH, 100); Anal. (C₁₆ H₁₅ N₃ O₃.HCl) C, H, N.

4-(3'-Bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P154

Yield 89.90%; mp 233.0-233.5° C.; ¹ H NMR (DMSO-d₆) d 10.08 (s, 1H,--NH), 9.38 (s, 1H, --OH), 8.40 (s, 1H, 2-H), 7.89 (d, 1H, J_(2'),6'=2.7 Hz, 2'-H), 7.75 (s, 1H, 5-H), 7.55 (dd, 1H, J_(5'),6' =9.0 Hz,J_(2'),6' =2.7 Hz, 6'-H), 7.14 (s, 1H, 8-H), 6.97 (d, 1H, J_(5'),6' =9.0Hz, 5'-H), 3.92 (s, 3H, --OCH₃), 3.90 (s, 3H, --OCH₃); IR (KBr) 3431,2841, 1624, 1498, 1423, 1244 cm⁻¹ ; GC/MS m/z 378 (M⁺ +2, 91), 377 (M⁺+1, 37), 376 (M⁺, 100), 360 (M⁺, 4), 298 (19), 282 (7); Anal. (C₁₆ H₁₄BrN₃ O₃.HCl) C, H, N.

4-(3'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline WHI-P180

Was synthesized as described previously (Narla et al., 1998, Clin.Cancer Res., 4:1405-1414.)

4-(Phenyl)-amino-6,7-dimethoxyquinazoline WHI-P258

Yield 88.26%; mp 258.0-260.0° C.; ¹ HNMR (DMSO-d₆) d 11.41 (s, 1H,--NH), 8.82 (s, 1H, 2-H), 8.32 (s, 1H, 5-H), 7.70-7.33 (m, 5H,2',3',4',5',6'-H), 7.36 (s, 1H, 8-H), 4.02 (s, 3H, --OCH₃), 4.00 (s, 3H,--OCH₃); IR (KBr) 2852, 1627, 1509, 1434, 1248 cm⁻¹ ; GC/MS m/z 282(M⁺+1, 11), 281 (M⁺, 55), 280 (M⁺ -1, 100), 264 (16), 207 (9); Anal. (C₁₆H₁₅ N₃ O₂) C, H, N.

Example 4 JAK-3 Expression in Mast Cells

Expression of JAK-3 in mast cells was studied.

Materials and Methods

Mice--Male C57BL/6 and Balb/c mice (6-8 weeks old) were purchased fromCharles River Laboratories (Wilmington, Mass.). Breeder pairs ofJAK3-null mice (Nosaka et. al., 1995) were obtained from Dr. J. Ihle(St. Jude Children's Research Hospital, Memphis, Tenn.). Animals werecaged in groups of five in a pathogen free environment in accordancewith the rules and regulations of U.S. Animal Welfare Act, and NationalInstitutes of Health (NIH). Animal care and the experimental procedureswere carried out in agreement with institutional guidelines.

Fetal bovine serum was obtained from Hyclone (Logan, Utah).Histopaque-1077, A23187, bovine serum albumin, toluidine blue, hydrogenperoxide, napthol AS-MX phosphate, fast blue RR, alcian blue, anti-humanIgE, and dimethyl sulphoxide (DMSO) were purchased from Sigma (St.Louis, Mo.). Leukotriene (LT) C₄ ELISA kits were from Cayman Company(Ann Arbor, Mich.). Histamine ELISA kits were purchased from Immunotech(Westbrook, Me.). The preparation of dinitrophenyl (DNP)-BSA (Wei et.al., 1986, J. Immunol., 137:1993-2000), monoclonal anti-DNP-IgE (Liu et.al., 1980, J. Immunol., 124:2728-2737) and FcεRIa chain antibody, 22E7(Riske et. al., 1991, J. Biol. Chem., 266:11245-11251) have beendescribed. Recombinant hSCF and IL-4 were purchased from Genzyme(Cambridge, Mass.). Alkaline phosphatase labeled anti-tryptase antibodywas purchased from Chemicon (Temecula, Calif.). Affinity purified JAK3and STAT5 antibodies were purchased from Quality Control Biochemicals(Hopkins, Mass.). Anti-phosphotyrosine monoclonal antibody (Mab) waspurchased from Upstate Biotechnology Inc. Human IgE was purchased fromCalbiochem (San Diego, Calif.).

Mast Cell Cultures

RBL-2H3 Cells

RBL-2H3 cells were a gift from Dr. Reuben P. Siraganian (Laboratory ofMicrobiology and Immunology, National Institute of Dental Research,National Institute of Health). The cells were maintained as monolayercultures in 75- or 150-cm² flask in Eagle's essential mediumsupplemented with 20% fetal calf serum (Hamawy et. al., 1995, CellularSignalling 7:535-544).

Mouse Mast Cells

Mast cells were cultured from the bone marrow specimens of JAK3-null(Jak3^(-/-)) and JAK3^(+/+) control mice in a medium supplemented with25% WEHI-3 cell supernatant for 3 weeks, as previously described(Malaviya et. al., 1993, J. Biol. Chem., 268:4939-4944; Malaviya et.al., 1994a, J. Immunol., 152:1907-1914). Cell density was adjusted to1×10⁵ cells/ml on a weekly basis. After 3 weeks, mast cells werecharacterized by staining with toluidine blue and alcian blue. Mastcells show metachromatic granules after staining with toluidine blue,whereas alcian blue specifically stains mast cell granules containingchrondroitin sulfate.

Human Mast Cells

Cells from human fetal livers (16 to 21 weeks of gestational age) werecultured for 5 weeks in the presence of 50 ng/ml rhSCF, 2 ng/ml rhIL-4(Xia et. al., 1997, J. Immunol., 159:2911-2921). Culture medium wasreplaced with fresh medium once a week for the first 2 weeks and twice aweek thereafter. At the end of the 5 weeks, the fetal liver derived cellcultures contained >70% mast cells, based on tryptase staining (Iraniet. al., 1989, J. Histochem. Cytochem, 37:1509-1515).

Confocol Microscopy

Staining of mast cells with primary and secondary antibodies followed byconfocal laser scanning microscopy was performed as previously describedin detail (Uckun et. al., 1998, Clin. Cancer Res., 4:901-912). Afterstaining with appropriate primary and secondary antibodies, cells werewashed three times to remove unbound antibody. DNA labeling wasperformed by incubation of coverslips with toto-3 (Molecular Probes,Eugene, Oreg.) for 10 min. Excessive dye was washed with PBS-0.1% tritonX-100. Cells were visualized under MRC 1024 Laser Scanning Microscopeafter mounting with Vectashield (Vector laboratories, Inc, Burlingame,Calif.). The anti-JAK3 (Witthuhn et. al., 1999, Lymphoma Leukemia, inpress) and anti-tubulin (clone B-5-1-2, Sigma Chemicals, St. Louis, Mo.)antibodies were used according to standard procedures (Uckun, et. al.,1998, Clin. Cancer Res., 4:901-912).

Immune Complex Kinase Assays

Sf21 (IPLB-SF21-AE) cells (Vassilev et. al., 1999, J. Biol. Chem,.274:1646-1656) derived from the ovarian tissue of the fall armywormSpodotera frugiperda, were obtained from Invitrogen and maintained at26-28° C. in Grace's insect cell medium supplemented with 10% FBS and1.0% antibiotic/antimycotic (GIBCO-BRL). Stock cells were maintained insuspension at 0.2-1.6×10⁶ /ml in 600 ml total culture volume in 1 LBellco spinner flasks at 60-90 rpm. Cell viability was maintained at95-100% as determined by trypan blue dye exclusion.

Sf21 cells were infected with a baculovirus expression vector for BTK,SYK, JAK1, JAK2, or JAK3, as previously reported (Mahajan et. al., 1999,J. Biol. Chem., 274:1646-1656). The human B-lineage leukemia cell lineNALM-6 and the EBV-transformed lymphoblastoid B-cell line KL-2 weremaintained in RPMI 1640 medium supplemented with 10% heat-inactivatedfetal bovine serum (Uckun et. al., 1996, Science, 273:1096-1100; Uckunet. al., 1995, Science, 267:886-891). Cells were harvested, lysed (10 mMTris pH 7.6, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 100mM Na₃ VO₄, 50 mg/ml phenylmethylsulfonyl fluoride, 10 mg/ml aprotonin,10 mg/ml leupeptin), and the kinases were immunoprecipitated from thelysates, as reported (Vassilev et. al., 1999, J. Biol. Chem.,724:1646-1656). Antibodies used for immunoprecipitations from insectcells are as follows: polyclonal rabbit anti-BTK serum, polyclonalrabbit anti-JAK1 (HR-785), cat# sc-277, polyclonal goat anti-JAK2(C-20-G), cat.# sc-294-G, polyclonal rabbit anti-JAK3 (C-21), cat #sc-513, polyclonal rabbit anti-SYK (C-21) cat# sc-929 (Santa CruzBiotechnology). Antibodies directed against BTK and LYN have beendescribed previously (Mahajan et. al., 1995, Mol. Cell. Biol.,15:5304-5311; Uckun et. al., 1996, Science, 273: 1096-1100; Uckun et.al., 1995, Science, 267:886-891; Uckun et. al., 1996a, J. Biol. Chem.,271:6396-6397.; Vassilev et. al., 1999, J. Biol. Chem., 274:1646-1656;Goodman et. al., 1998, J. Biol. Chem., 273:17742-17748). Immune-complexkinase assays were conducted as described in these references. Kinaseassays were performed following a 1 hour exposure of theimmunoprecipitated tyrosine kinases to the test compounds, as describedin detail elsewhere (Mahajan et. al., 1995, Mol. Cell. Biol.,15:5304-5311; Uckun et. al., 1996, Science, 273:1096-1100). Theimmunoprecipitates were subjected to Western blot analysis as previouslydescribed (Uckun et. al., 1996, Science, 273:1096-1100; Uckun et. al.,1996a, J. Biol. Chem,. 271:6396-6397; Vassilev et. al., 1999, J. Biol.Chem., 274:1646-1656).

For insulin receptor kinase (IRK) assays, HepG2 human hepatoma cellsgrown to approximately 80% confluency were washed once with serum-freeDMEM and starved for 3 hours at 37° in a CO₂ incubator. Subsequently,cells were stimulated with insulin (Eli Lilly, cat# CP-410; 10units/ml/10×10⁶ cells) for 10 minutes at room temperature. Followingthis IRK activation step, cells were washed once with serum free medium,lysed in NP-40 buffer and IRK was immunoprecipitated from the lysateswith an anti-IRb antibody (Santa Cruz, Cat.# sc-711, polyclonal IgG).Prior to performing the immune complex kinase assays, the beads wereequilibrated with the kinase buffer (30 mM Hepes pH 7.4, 30 mM NaCl, 8mM MgCl₂, 4 mM MnCl₂). LYN was immunoprecipitated from whole celllysates of NALM-6 human leukemia cells as previously reported (Uckun et.al., 1995, Science, 267:886-891).

In JAK3 immune complex kinase assays (Goodman et. al., 1998, J. Biol.Chem., 273:17742-17748; Witthuhn et. al., 1999, Lymphoma Leukemia, inpress"; Mahajan, et. al., 1999, J. Biol. Chem., in press). KL-2EBV-transformed human lymphoblastoid B cells (native JAK3 kinase assays)or insect ovary cells (recombinant JAK3 kinase assays) were lysed withNP-40 lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 1% NP-40,100 μM sodium orthovanadate, 100 μM sodium molybdate, 8 μg/ml aprotinin,5 μg/ml leupeptin, and 500 μM PMSF) and centrifuged 10 minutes at13000×g to remove insoluble material. Samples were immunoprecipitatedwith antisera prepared against JAK3. The antisera were diluted andimmune complexes collected by incubation with 15 μl protein A Sepharose.After 4 washes with NP-40 lysis buffer, the protein A Sepharose beadswere washed once in kinase buffer (20 mM MOPS, pH 7.0, 10 mM MgCl₂) andresuspended in the same buffer. Reactions were initiated by the additionof 25 μCi γ[³² P] ATP (5000 Ci/mMole) and unlabeled ATP to a finalconcentration of 5 μM. Reactions were terminated by boiling for 4 min inSDS sample buffer. Samples were run on 9.5% SDS polyacrylamide gels andlabeled proteins will be detected by autoradiography. Followingelectrophoresis, kinase gels were dried onto Whatman 3M filter paper andsubjected to phosphoimaging on a Molecular Imager (Bio-Rad, Hercules,Calif.) as well as autoradiography on film. For each drug concentration,a kinase activity index (KA) was determined by comparing the kinaseactivity in phosphorimager units (PIU) to that of the baseline sample.In some experiments, cold kinase assays were performed, as described(Uckun et. al., 1998, Clin. Cancer Res., 4:901-912).

Electrophoretic Mobility Shift Assays (EMSAs)

EMSAs were performed to examine the effects of dimethoxyquinazolinecompounds on cytokine-induced STAT activation (Goodman et. al., 1998).Specifically, 32Dc11/IL2Rβ cells (gift from James Ihle, St. JudeChildren's Research Hospital) were exposed at 8×10⁶ /ml in RPMIsupplemented with FBS to WHI-P131, WHI-P154, or the control compoundWHI-P132 at a final concentration of 10 μg/ml in 1% DMSO for 1 hour andsubsequently stimulated with IL2 or IL3 as indicated in the text. Cellswere collected after 15 minutes and resuspended in lysis buffer (100 mMTris-HCl pH 8.0, 0.5% NP-40, 10% glycerol, 100 mM EDTA, 0.1 mM NaVO3, 50mM NaF, 150 mM NaCl, 1 mM DTT, 3 μg/ml Aprotinin, 2 μg/ml Pepstatin A, 1μg/ml Leupeptin and 0.2 mM PMSF). Lysates were precleared bycentrifugation for 30 min. Cell extracts (approximately 10 μg) wereincubated with 2 μg of poly(dI-dC) for 30 minutes, followed by a 30minutes incubation with 1 ng of poly nucleotide kinase-³² P labeleddouble stranded DNA oligonucleotide representing the IRF-1 STAT DNAbinding sequence (Santa Cruz Biotechnology, Santa Cruz, Calif.). Sampleswere resolved by non-denaturing PAGE and visualized by autoradiography.

Stimulation of Mast Cells

RBL-2H3 cells and bone marrow mast cells (BMMC) cultured from the bonemarrow cells of JAK3^(-/-) or JAK3^(+/+) mice were sensitized withmonoclonal anti-DNP IgE antibody (0.24 mg/ml) for 1 h at 37° C. in a48-well tissue culture plate. RBL-2H3 cells were allowed to adhere tothe plate, whereas BMMC were used in suspension. Unbound IgE was removedby washing the cells with phosphate buffered saline. After washing theBMMC were re-suspended in RPMI-hepes buffer whereas PIPES-bufferedsaline containing 1 mM calcium chloride was added to the monolayers ofthe RBL-2H3 cells. The cells were challenged with 20 ng/ml DNP-BSA for30 minutes at 37° C. The plate was centrifuged at 200 g for 10 minutesat 4° C. Supernatants were removed and saved. The RBL-2H3 cell pelletswere washed with phosphate buffered saline and solubilized in PIPESbuffered saline containing 0.1% Triton X-100.

Fetal liver derived human mast cells were resuspended in tyrode buffercontaining calcium and magnesium and challenged with anti-FcεRI antibody22E7 for 15 minutes. In some experiments fetal liver derived human mastcells were re-suspended in culture medium at a cell density of 5×10⁶ /mland sensitized with IgE (150 μg/ml) for 3 hours at 4° C. Aftersensitization the cells were washed with tyrode buffer containingcalcium and magnesium and challenged with mouse monoclonal anti-humanIgE (40 μg/ml) for 30 minutes at 37° C. To study the effects of the testcompounds, mast cells were incubated with WHI-P131, WHI-P154, WHI-P111or WHI-P112 at the indicated concentrations or vehicle for 1 hour priorto challenge.

Mediator Release Assays

Histamine content in cell free supernatants and in the solubilized cellpellets was estimated using a commercially available enzyme immunoassay(Malaviya et. al., 1996a, J. Invest. Dermatol, 106:785-789). Leukotriene(LT) C₄ levels were estimated in cell free supernatants by immunoassay(Malaviya et. al., 1993, J. Biol. Chem., 268:4939-4944). TNFα levelswere estimated in cell free supernatants using a standard cytotoxicityassay (Malaviya et. al., 1996, Nature, 381:77-80). In RBL-2H3 cells,β-hexosaminidase release was estimated in cell free supernatants andTriton X-100 solubilized pellets, as described (Ozawa et. al., 1993, J.Biol. Chem., 268:1749-1756). Tryptase levels were quantitated in cellfree supernatants and pellets of fetal liver derived human mast cells ashas been described in detail (Xia et. al., 1997, J. Immunol.,159:2911-2921).

Proliferation Assay

The proliferative responses of mast cells to SCF were determined usingthe MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)assay (Boehringer Mannheim Corp., Indianapolis, Ind.) (Uckun et. al.,1998, Clin. Cancer Res., 4:901-912). Briefly wild-type and JAK3 nullBMMC were cultured in 96-well tissue culture plates at a cell density of0.1×10⁵ cells/ml in 100 μl media in the presence and absence of 100ng/ml SCF for 96 hours at 37° C. To each well 10 μl of MTT (finalconcentration, 0.5 mg/ml) was added, and the plates were incubated at37° C. for 4 hours to allow formazan crystals to form by reacting withmetabolically active cells. The formazan crystals were solubilizedovernight at 37° C. in a solution containing 10% SDS in 0.01N-HCl. Theabsorbance of each well was measured in a microplate reader (Labsystems)at a wave length of 540 nm.

IgE Binding Assay

RBL-2H3 cells were incubated of 0.24 mg/ml monoclonal anti-DNP IgEantibody for 1 hour at 37° C. The cells were washed thoroughly threetimes with saline containing 1% BSA and labeled with 10 μg/mlanti-IgE-FITC (PharMingan Laboratories) for 30 minutes at 4° C.Following incubation the cells were washed were analyzed by flowcytometry.

Calcium Measurement

Calcium mobilization assay was performed as described earlier (Zhu et.al., 1998, Clin. Cancer Res., 4:2967-2976). RBL-2H3 cells were loadedwith Fluo-3 and stimulated with DNP-BSA in presence and absence ofWHI-P131 as described above. Calcium response was measured by an calciumimaging device (Universal Imaging Co., West Chester, Pa.) mounted onto ainverted microscope. The exitation wavelength was 485 nm and theemission wavelength for detection was 535 nm. The fluorescent image ofan individual cell was acquired by a CCD72 video camera (Dage-MTI Inc.,Michigan City, Ill.) at the speed of 1 frame/s and digitized bycomputer.

Immune-Complex Kinase Assays and Western Blot Analyses of Mast CellLysates

RBL-2H3 cells were stimulated as described above in the presence andabsence of WHI-P131 for the indicated times. Cells were harvested, lysed(10 mM Tris pH 7.6, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 50 mMNaF, 100 mM Na₃ VO₄, 50 mg/ml phenylmethylsulfonyl fluoride, 10 mg/mlaprotonin, 10 mg/ml leupeptin), and the kinases were immunoprecipitatedfrom the lysates, as reported (Uckun et. al., 1995, Science,267:886-891; Uckun et. al., 1996, Science, 273:1096-1100). Antibodiesdirected against JAK3, STAT5, and SYK used for immunoprecipitations havebeen described previously (Uckun et. al., 1995, Science, 267:886-891;Uckun et. al., 1996, Science, 273:1096-1100; Witthuhn et. al., 1999,Lymphoma Leukemia, in press"; Mahajan et. al., 1999, J. Biol. Chem., inpress). Immunoprecipitations, and immunoblotting using the ECLchemiluminescence detection system (Amersham Life Sciences) wereconducted as described previously (Uckun et. al., 1996, Science,273:1096-1100).

Anaphylaxis Models

In order to examine the effect of WHI-P131 on passive cutaneousanaphylaxis in mice, dorsal sides of the ears of BALB/c mice wereinjected intradermally with 20 ng of DNP-IgE (left ears) or PBS (rightears) in 20 μL volume using a 30-gauge needle, as previously described(Miyajima et. al., 1997, J. Clin. Invest., 99:901-914). After 20 hours,mice were treated with WHI-P131 (10 or 25 mg/kg i.p.) twice at 1 hourintervals prior to the antigen challenge. Control mice were treated withan equal volume of vehicle. Thirty minutes after the last dose ofWHI-P131 or vehicle, mice were challenged with 100 μg antigen (DNP-BSA)in 200 μl 2% Evans blue dye intravenously. Mice were sacrificed bycervical dislocation 30 minutes after the antigen challenge. Forquantitation of Evans blue dye extravasation as a measure ofanaphylaxis-associated vascular hyperpermeability, 8 mm skin specimenswere removed from the ears of mice, minced in 2 ml formamide andincubated at 80° C. for 2 hours in water bath to extract the dye. Theabsorbance was read at 620 nM. The data were expressed as plasmaexudation indices (i.e., times increase in optical density over PBStreated ears at 620 nm).

To induce passive systemic anaphylaxis, BALB/c mice were sensitized with50 μg DNP-IgE intravenously. At 24 hours DNP-BSA (2 mg) was administeredintravenously with 0.5% Evans blue (200 μl). For assessment of vascularleak, animals were sacrificed 30 minutes after the antigen challenge andtheir foot pads were examined for blue coloration. Histamine levels inplasma were measured 5 minutes after the antigen challenge. To this end,blood samples were obtained from the ocular venous plexus byretroorbital venupuncture and histamine levels were determined by ELISAusing a commercial kit (Immunotech, West Brook, Me., Malaviya et. al.,1996, Nature, 381:77-80). For histopathologic evaluation of tissue mastcell degranulation, ears of mice were removed 1 hour after the DNP-BSAinjection and fixed in 10 percent buffered formalin. Processed thinsections (3-5 μm) were stained with Avidin-FITC (6.25 μg/ml) for 2hours, washed with PBS to remove unbound dye and then mounted inbuffered glycerol, 30 mM triethylenediamine, pH 8.6 (Malaviya et. al.,1994, J. Clin. Invest., 93:1645-1653). In the murine model for antigeninduced active anaphylaxis, mice were sensitized with 2 mg BSA in 200 μlaluminum hydroxide gel (Reheis Inc., Berkeley, N.J.). Ten days lateranaphylactic shock was induced by the i.v. injection of the animals with200 μg BSA.

Toxicity Studies in Mice

The toxicity profile of WHI-P131 in mice was examined, as previouslyreported for other new agents (Uckun et. al., 1995, Science,267:886-891; Uckun et. al., 1998, Clin. Cancer Res., 4:901-912). FemaleBALB/c mice were used and monitored daily for lethargy, cleanliness andmorbidity. At the time of death, necropsies were performed and the toxiceffects of WHI-P131 administration were assessed. For histopathologicstudies, tissues were fixed in 10% neutral buffered formalin,dehydrated, and embedded in paraffin by routine methods. Glass slideswith affixed 6 micron tissue sections were prepared and stained withHemotoxylin and Eosin (H&E). Female BALB/c mice were administered ani.p. bolus injection of WHI-P131 in 0.2 ml PBS supplemented with 10%DMSO, or 0.2 ml PBS supplemented with 10% DMSO alone (control mice). Nosedation or anesthesia was used throughout the treatment period. Micewere monitored daily for mortality for determination of the day 30 LD₅₀values. Mice surviving until the end of the 30 days monitoring weresacrificed and the tissues were immediately collected from randomlyselected mice, and preserved in 10% neutral buffered formalin. Standardtissues collected for histologic evaluation included: bone, bone marrow,brain, cecum, heart, kidney, large intestine, liver, lung, lymph node,ovary, pancreas, skeletal muscle, skin, small intestine, spleen,stomach, thymus, thyroid gland, urinary bladder, and uterus (asavailable).

Pharmacokinetic Studies

In pharmacokinetic studies, mice were injected either intravenously(i.v.) via the tail vein or intraperitoneally (i.p.) with a bolus doseof 300 μg/mouse (˜12.5 mg/kg=34 μmols/kg) of WHI-P131. Blood sampleswere obtained from the ocular venous plexus by retroorbital venupunctureprior to and at 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30minutes, 45 minutes and 1 hour, 2 hours, 4 hours and 8 hours afteradministration of WHI-P131. All collected blood samples were heparinizedand centrifuged at 7,000×g for 10 minutes in a microcentrifuge to obtainplasma. The plasma samples were stored at -20° C. until analysis.Aliquots of plasma were used for extraction and HPLC analysis.Pharmacokinetic modeling and pharmacokinetic parameter calculations werecarried out using the pharmacokinetics software, WinNonline Program,Version 1.1. (Scientific Consulting Inc., Cary, N.C.). Concentrationdata were weighted by 1/concentration. An appropriate pharmacokineticmodel was chosen on the basis of lowest weighted squared residuals,lowest Schwartz criterion (SC), lowest Akaike's Information Criterion(AIC) value, lowest standard errors of the fitted parameters, anddispersion of the residuals. The elimination half-life was estimated bylinear regression analysis of the terminal phase of the plasmaconcentration profile. The area under the concentration time curve (AUC)was calculated by the trapezoidal rule between first (0 h) and lastsampling time plus C/k, where C is the concentration of last samplingand k is the elimination rate constant. Systemic clearance (CL) wasdetermined by dividing the dose by the AUC. The apparent volume ofdistribution at steady-state was calculated using the followingequation, V_(ss) =Dose·AUMC/(AUC)². Bioavailability (F) was estimatedusing the equation F(%)=AUC_(ip) ×Dose_(iv) /AUC_(iv) ×Dose_(ip).

HPLC Analysis of Plasma WHI-P131 Levels

A highly sensitive quantitative HPLC detection method (Chen et. al.,1998, J. Chromatography B (Biomedical sciences), in press) was used todetermine the pharmocokinetics of WHI-P131. In brief, the HPLC systemconsisted of a Hewlett Packard (HP) series 1100 equipped with anautomated electronic degasser, a quaternary pump, an autosampler, anautomatic thermostatic column compartment, diode array detector and acomputer with a Chemstation software program for data analysis. A 250×4mm Lichrospher 100, RP-18 (5 μm) analytical column and a 4×4 mmLichrospher 100, RP-1 8 (5 μm) guard column were obtained from HewlettPackard Inc. (San Fernando, Calif.). Acetonitrile/water containing 0.1%of trifluoroacetic acid (TFA) and 0.1% triethylamine (TEA) (28:72, v/v)was used as the mobile phase. The wavelength of detection was set at 340nm. Peak width, response time and slit were set at >0.03 minutes, 0.5seconds and 8 nm, respectively. For determination of WHI-P131 levels, 10μL of internal standard WHI-P154 (50 μM) was added to a 100 μL plasmasample. For extraction, 7 ml chloroform was then added to the plasmasample, and the mixture was vortexed thoroughly for 3 minutes. Followingcentrifugation (300×g, 5 minutes), the aqueous layer was frozen usingacetone/dry ice and the organic phase was transferred into a clean testtube. The chloroform extracts were dried under a slow steady stream ofnitrogen. The residue was reconstituted in 100 μL of methanol:water(9:1) and 50 μL aliquot of this solution was used for HPLC analysis.Under the described chromatographic separation conditions, the retentiontimes for WHI-P131 and WHI-P154 were 5.1 minutes and 9.5 minutes,respectively. At the retention time, WHI-P131 and its internal standardWHI-P154 were eluted without any interference peaks from the blankplasma.

Example 5 Expression and IgE Receptor/FcεRI-mediated Activation of JanusKinase 3 in Mast Cells

As shown in FIG. 4A, JAK3 is abundantly expressed in RBL-2H3 mast cells.This finding prompted us to examine the potential involvement of JAK3 inIgE receptor/FcεRI-mediated mast cell activation. Crosslinking of theIgE receptors on RBL-2H3 mast cells that were previously sensitized by amonoclonal anti-dinitrophenyl (DNP)-IgE antibody, with the specificantigen DNP-BSA resulted in rapid activation of JAK3 (FIG. 4B).

To elucidate the role of JAK3 in IgE receptor/FcεRI mediated mast cellresponses, we cultured mast cells from the bone marrows of wild-type andJAK3-null mice (Jak3^(-/-)) that were generated by targeted disruptionof Jak3 gene in embryonic stem cells (Nosaka et. al., 1995, Science,270:800-802). Similar numbers of mast cells (range: 3.5-3.9×10⁵ per 10⁶nucleated bone marrow cells) were obtained from bone marrow specimens ofJAK3^(+/+) and Jak3^(-/-) mice after 3 weeks of culture in mediumsupplemented with WEHI-3 cell supernatants (25% v/v). JAK3^(-/-) mastcells showed typical mast cell staining characteristics towards alcianblue and toluidine blue (data not shown). When we compared the FcεRIreceptor expression between JAK3^(+/+) and JAK³ ^(-/-) mast cells wefound that JAK3^(+/+) and JAK3^(-/-) mast cells express similar levelsof IgE receptor/FcεRI (FIG. 5A). Thus, JAK3 deficiency does not affectIgE receptor expression on mast cells. Fully differentiated mast cellsexpress functional c-kit receptors and activation of c-kit receptor byits ligand, stem cell factor (SCF), triggers proliferation of mast cells(Dvorak et. al., 1994, Am. J. Pathol. 144:160-170). We compared theproliferative responses of JAK3 versus JAK3^(+/+) mast cells to SCFusing MTT assays. JAK3^(+/+) or JAK3^(-/-) mast cells at a cell densityof 0.1×10⁵ cells/ml, were incubated in the presence and absence of 100ng/ml SCF for 96 hours at 37° C. At the end of the SCF incubation,virtually identical cell counts were obtained from cultures ofJAK3^(+/+) and JAK3^(-/-) mast cells (FIG. 5B). Thus, JAK3 deficiencydoes not affect the in vitro proliferative responses of mast cells toSCF.

To further evaluate the potential role of JAK3 in mast cell responses,we next compared the IgE receptor/FcεRI mediated release ofinflammatory/allergic mediators from JAK3^(+/+) and JAK3^(-/-) mastcells. Mast cells cultured from the bone marrows of JAK3^(+/+) andJAK3^(-/-) mice were sensitized with anti-DNP monoclonal IgE andchallenged with DNP-BSA. The release of preformed granule-associatedhistamine, a marker for mast cell degranulation, was measured bycomparing the histamine contents of the in cell free supernatants andcell pellets. The release of the arachidonic acid metabolite LTC₄, whichserves as a marker for release of newly synthesized mediators, wasestimated by determining the LTC₄ levels in cell free supernatants.Although the cellular histamine contents of JAK3^(-/-) mast cells andJAK3^(+/+) mast cells were identical, JAK3^(-/-) mast cells releasedapproximately half of the amount of histamine released by JAK3^(+/+)mast cells (FIG. 5C). LTC₄ release (FIG. 5D) after IgE receptor/FcεRIcrosslinking was also substantially reduced with JAK3^(-/-) mast cells.These results indicate that JAK3 plays an important role in IgEreceptor/FcεRI-mediated mast cell responses. In agreement with these invitro results, none of the BSA-sensitized Jak3^(-/-) mice (N=3) showedan adverse reaction to a rechallenge with 200 μg BSA injectedintraperitoneally in our active systemic anaphylaxis (ASA) model (Amirand English, 1991), whereas all BSA-sensitized JAK3^(+/+) mice (N=5)developed a severe anaphylactic reaction within 10 minutes after BSArechallenge under the same experimental conditions (data not shown).Taken together, these experiments provided unprecedented evidence thatthe IgE receptor/FcεRI-mediated activation of mast cells triggers abiochemical JAK3-activation signal which is important for pleiotropicbiological mast cell responses to IgE receptor/FcεRI-engagement andhence for mast cell-mediated hypersensitivity reactions.

Example 6 Effects of Dimethoxy Quinozalines on JAK-3 Activity

We first used immune complex kinase assays to compare the effects of thesynthesized dimethoxyquinazoline compounds on the enzymatic activity ofhuman JAK3 immunoprecipitated from the KL2 EBV-transformed humanlymphoblastoid B cell line. WHI-P131, WHI-P154, and WHI-P97, which hadvery similar estimated K_(i) values ranging from 0.6 μM to 2.3 μM andwere predicted to show significant JAK3 inhibitory activity atmicromolar concentrations (which was not the case for the othercompounds which had estimated K_(i) values ranging from 25 μM to 72 μM),inhibited JAK3 in dose-dependent fashion. The measured IC₅₀ values were9.1 μM for WHI-P131, 11.0 μM for WHI-P97, and 27.9 μM for WHI-P154,but >300 μM for all the other dimethoxyquinazoline compounds (Table 1).WHI-P131 and WHI-P154 were also tested against recombinant murine JAK3expressed in a baculovirus vector expression system and inhibited JAK3in a dose-dependent fashion with an IC₅₀ value of 23.2 μg/ml (˜78 mM,FIG. 6A) and 48.1 μg/ml (˜128 μM, FIG. 6B), respectively. The ability ofWHI-P131 and WHI-P154 to inhibit recombinant JAK3 was confirmed in fourindependent experiments. These kinase assay results are consistent withour modeling studies described above.

Importantly, WHI-P131 and WHI-P154 did not exhibit any detectableinhibitory activity against recombinant JAK1 or JAK2 in immune complexkinase assays (FIGS. 6C & D). Electrophoretic Mobility Shift Assays(EMSAs) were also performed to confirm the JAK3 specificity of thesedimethoxyquinazoline compounds by examining their effects oncytokine-induced STAT activation in 32Dc11/IL2Rβ cells. As shown in FIG.6E, both WHI-P131 (10 μg/ml=33.6 μM) and WHI-P154 (10 μg/ml=26.6 μM)(but not the control compound WHI-P132, 10 μg/ml=33.6 μM) inhibitedJAK3-dependent STAT activation after stimulation with IL-2, but they didnot affect JAK1/JAK2-dependent STAT activation after stimulation withIL-3. Modeling studies suggest that this exquisite JAK3 specificitycould in part be due to an alanine residue (Ala966) which is present inthe catalytic site of JAK3 but changes to glycine in JAK1 and JAK2. Thisalanine group which is positioned near the phenyl ring of the bounddimethoxyquinazoline compounds can provide greater hydrophobic contactwith the phenyl group and thus contribute to higher affinity relative tothe smaller glycine residue in this region of the binding site in JAK1and JAK2.

Example 7 Specificity of JAK-3 Inhibitors

Compound WHI-P131 was selected for further experiments designed toexamine the sensitivity of non-Janus family protein tyrosine kinases tothis novel dimethoxyquinazoline class of JAK3 inhibitors. The inhibitoryactivity of WHI-P131 against JAK3 was specific since it did not affectthe enzymatic activity of other protein tyrosine kinases, including theZAP/SYK family tyrosine kinase SYK, TEC family tyrosine kinase BTK, SRCfamily tyrosine kinase LYN, and receptor family tyrosine kinase IRK evenat concentrations as high as 350 μM (FIG. 7).

A structural analysis of these PTK was performed using the crystalstructures of HCK (Sicheri et. al., 1997) (which served as a homologymodel for LYN) IRK (Hubbard, 1997) and constructed homology models ofJAK3, BTK, and SYK. This analysis revealed some nonconserved residueslocated in the catalytic binding site of the different tyrosine kinaseswhich may contribute to the specificity of WHI-P131 (FIG. 3). One suchresidue which is located closest to the docked inhibitors is Ala966 inJAK3 (shown in region E in FIG. 5) which may provide the most favorablemolecular surface contact with the hydrophobic phenyl ring of WHI-P131.The fact that WHI-P131 did not inhibit LYN, even though LYN contains theAla residue conserved in JAK3 (Ala966), suggests that other factors(residue differences) contribute to this selectivity. Other nonconservedresidues in the catalytic site of tyrosine kinases are shown in regionsA to F (FIG. 5A). All of these differences in residues, especiallyresidues which directly contact the bound inhibitor, may play animportant role in the observed specificity of WHI-P131 for JAK3.

Example 8 Effects of JAK3 Inhibitors on In Vitro Mast Cell Responses

Treatment of the rat mucosal mast cell line RBL-2H3 with theJAK3-specific tyrosine kinase inhibitor WHI-P131 abrogated JAK3activation after IgE receptor crosslinking (FIGS. 8A & B). Notably,WHI-P131 did not prevent SYK activation in RBL-2H3 mast cells after IgEreceptor/FcεRI crosslinking (FIGS. 8C & D). Therefore, any biologicconsequences of JAK3 inhibition in WHI-P131-treated mast cells can notbe attributed to impaired SYK activation.

In a systematic effort aimed at elucidating the biologic consequences ofJAK3 inhibition in mast cells, we first sought to determine if the JAK3inhibitor WHI-P131 could prevent the IgE receptor/FcεRI-mediatedactivation of mast cells. Since the IgE receptor/FcεRI-mediatedactivation of mast cells results in a distinct morphologictransformation with marked cell spreading due to membrane ruffling,microtubule formation and actin polymerization (Apgar, 1997), weevaluated the effects of WHI-P131 on the activation-associatedtransformation of shape and surface topography of RBL-2H3 mast cellsusing confocal laser scanning microscopy (Ghosh et. al., 1998). The vastmajority (95%) of unstimulated RBL-2H3 mast cells exhibited a spindleshape with arborized extensions and longitudinally oriented bundles ofmicrotubules (FIG. 9A). Activation of RBL-2H3 mast cells by crosslinkingtheir IgE receptors/FcεRI using IgE/antigen induced a dramatic cellspreading response and 93% of cells assumed a flattened shape with ageneralized microtubule organization throughout their cytoplasm (FIG.9B). A two hour incubation with the JAK3 inhibitor WHI-P131 (but not theunsubstituted parent dimethoxyquinazoline compound WHI-P258, which lacksJAK3 inhibitory activity) at a concentration of 30 μM prevented theIgE/antigen-induced mast cell activation, as evidenced by markedlyreduced spreading (23% flattened; 58% spindle shaped) and microtubuleorganization (FIGS. 9 C-F). In contrast to WHI-P131, thebromo-substituted control dimethoxyquinazoline compound WHI-112, whichdoes not inhibit JAK3, was unable to produce any significant effects onantigen induced mast cell spreading or microtubule organization (FIGS.9G & H). In parallel, we tested the effect of the compounds on theviability of RBL-2H3 cells (as assessed by trypan blue dye exclusiontest) under these experimental conditions and found that they do notaffect cell viability up to 300 μM concentrations (data not shown).

Example 9 Effect on Calcium Mobilization

Since calcium influx is one of the earliest events in IgEreceptor/FcεRI-mediated mast cell activation (Millard et. al., 1989;Hamawy et. al., 1995), we also investigated whether Ca²⁺ mobilization isaffected by the JAK3 inhibitor WHI-P131. RBL-2H3 cells were sensitizedwith IgE and loaded with Fluo-3 prior to stimulation with DNP-BSA. Theintracellular calcium ion concentration reached a maximum within 2-3 minafter stimulation (FIG. 10) which is consistent with previous findings(Millard et. al., 1989; Wong et. al., 1992). In contrast, when RBL-2H3cells were stimulated with DNP-BSA in presence of 30 μM WHI-P131, thecalcium response was abrogated (FIG. 10). Thus, JAK3 is essential forIgE receptor/FcεRI-mediated activation and calcium mobilization in mastcells. In contrast, ionomycin induced nonspecific mast cell calciumresponses were not affected by WHI-P131 (FIG. 10).

Example 10 Mast Cell Degranulation and Mediator Release

In order to further examine the role of JAK3 in IgEreceptor/FcεRI-mediated mast cell activation and degranulation, we nextassessed the effects of the JAK3 inhibitors WHI-P131 and WHI-P154 onmast cell degranulation and mediator release induced by IgE/antigen.WHI-P111 and WHI-P112, which do not inhibit JAK3, were included ascontrols. RBL-2H3 mast cells were preincubated with increasingconcentrations of the test compounds or vehicle for 1 h before challengewith antigen (DNP-BSA). Stimulation of RBL-2H3 mast cells usingIgE/antigen resulted in release of significant amounts ofβ-hexosaminidase (45.1±3.1% of total cellular content), LTC₄ (11.3±1.3pg/10⁶ cells), and TNFa (160±33.0 pg/10⁶ cells). Notably, both JAK3inhibitors, WHI-P131 and WHI-P154, prevented mast cell degranulation andrelease of preformed granule-associated β-hexosaminidase (FIG. 11A) aswell as release of the newly synthesized arachidonic acid metaboliteLTC₄ (FIG. 11B) and the proinflammatory cytokine TNFa (FIG. 11C) in adose-dependent fashion with near to complete inhibition at ≧30 μM.Unlike these JAK3 inhibitors, the control dimethoxyquinazolinederivatives WHI-P111 and WHI-P112 lacking JAK3 inhibitory activity didnot inhibit mast cell degranulation or mediator release after IgEreceptor/FcεRI crosslinking (FIGS. 11A-C).

The effect of WHI-P131 on RBL-2H3 was dependent on the presence of thedrug and caused by an irreversible cytopathic effect of the drugpreincubation because when the bulk of WHI-P131 was removed by repeatedwashing of the cells prior to antigen challenge, the inhibitory effectof the drug was significantly reduced (data not shown). Similar to theseresults obtained using the rat mast cell line RBL-2H3, treatment withthe JAK3 inhibitor WHI-P131 markedly reduced IgE receptor/FcεRI-mediatedrelease of histamine and LTC₄ from mouse bone marrow mast cells as well(data not shown).

The effects of WHI-P131 on IgE receptor/FcεRI mediated degranulation andmediator release from human mast cells was next examined. Fetal liverderived human mast cells were cultured in presence of SCF and IL-4 for 5weeks. IgE-sensitized human mast cells were exposed to vehicle orincreasing concentrations of WHI-P131 for 30 minutes. Human mast cellsstore the mast cell specific protease β-tryptase in their secretorygranules (FIG. 12A) and the release of β-tryptase by degranulation is aspecific marker for human mast cell activation (Hogan and Schwartz,1997, Methods, 13:43-52). The FcεRI receptors of fetal liver derivedhuman mast cells were crosslinked with anti-IgE and the resulting mastcell degranulation (i.e., β-tryptase) (Xia et. al., 1997, J. Immunol.,159:2911-2921) and LTC₄ release (Malaviya et. al., 1993, J. Biol. Chem.,268:4939-4944) were quantitated. WHI-P131 inhibited the release ofβ-tryptase (FIG. 12B) as well as LTC₄ (FIG. 12C) from IgE/antigenstimulated human mast cells in a dose-dependent fashion.

In similar experiments, WHI-P180 effectively inhibited IgE/antigeninduced mast cell degranulation, as measured by β-hexasaminidaserelease, as well as secretion of newly synthesized mediators (LTC₄release) WHI-P180 was much more potent than the unsubstituteddimethoxyquinazolines WHI-P258. (Chen et al., 1999, Pharmac. Res.,16:117-122)

Taken together, these in vitro JAK3 inhibitor studies providedbiochemical evidence confirming and extending the genetic evidenceobtained using Jak3^(-/-) mast cells that JAK3 in mast cells is a keyregulator of IgE receptor/FcεRI-mediated responses. The ability of theJAK3 inhibitor WHI-P131 to inhibit mast cell degranulation as well asmediator release after IgE receptor/FcεRI crosslinking prompted us tofurther evaluate the potential of this compound as an anti-allergicagent.

Example 11 Effects of JAK3 Inhibitors on In Vivo Mast Cell Responses

WHI-P131 was not toxic to mice at intraperitoneal single bolus dosesranging from 0.5 mg/kg to 250 mg/kg. None of the 50 mice treated withWHI-P131 experienced side effects or died of toxicity during the 30 dayobservation period. In particular, we observed no hematologic sideeffects such as neutropenia, lymphopenia, or anemia at the tested doselevels. No histopathologic lesions were found in the organs of WHI-P131treated mice that were electively killed at 30 days and there was nobone marrow hypoplasia or lymphoid cell depletion in spleen and lymphnodes. Thus, the maximum tolerated dose (MTD) of WHI-P131 was notreached at 250 mg/kg. We next examined the pharmacokinetic features ofWHI-P131 in mice. A two-compartment pharmacokinetic model was fit to thepharmacokinetics data obtained following the intravenous (i.v.) (FIG.13A) or intraperitoneal (i.p.) (FIG. 13B) administration of a singlenon-toxic 12.5 mg/kg bolus dose of WHI-P131. The estimated maximumplasma concentrations (C_(max)) of WHI-P131 were 85.6 μM after i.v.administration and 57.7 μM after i.p. administration, which are higherthan the target effective concentration of 30 μM, at which WHI-P131abrogates mast cell responses in vitro. WHI-P131 demonstrated rapidabsorption after i.p. administration (estimated bioavailability: 94.4%)with an absorption half-life of 0.10 h, and the time to reach maximumplasma WHI-P131 concentration was 0.17 hours. WHI-P131 also had a rapidelimination rate with a β-half-life of 2.1 hours after i.v.administration and 1.9 hour after i.p. administration.

Example 12 Murine Models of Anaphylaxis

Taken together, these studies prompted the hypothesis that effectivemast cell inhibitory plasma concentrations of WHI-P131 can be achievedin vivo in mice receiving non-toxic doses of this potent JAK3 inhibitor.To test this hypothesis, the effects of WHI-P131 in murine models ofanaphylaxis were determined. Increased vascular permeability induced bymast cell mediators, such as histamine and leukotrienes, is a hallmarkof anaphylaxis (Oettgen et. al., 1994, Nature, 370:367-370; Miyajima et.al., 1997, J. Clin. Invest., 99:901-914).

Therefore, the effect of the JAK3 inhibitor WHI-P131 on vascularpermeability was first examined in a well-characterized murine model ofpassive cutaneous anaphylaxis (Miyajima et. al., 1997, J. Clin. Invest.,99:901-914). WHI-P131 inhibited the IgE/antigen induced plasmaexudation, as measured by extravasation of systemically administeredEvan's blue dye, in mice that had been presensitized with antigenspecific IgE by 70% at the 25 mg/kg nontoxic dose level (FIG. 14A).

Next, the effect of WHI-P131 on passive systemic anaphylaxis was studiedin mice (Amir and English, 1991, Eur. J. Pharmacol., 203:125-127;Oettgen et. al., 1994, Nature, 370:367-370; Miyajima et. al., 1997, J.Clin. Invest., 99:901-914). Mice were sensitized intravenously with 50μg anti DNP-IgE. Twenty four hours later, drug or vehicle-treatedanimals were challenged with 2 mg DNP-BSA systemically in presence of0.5% Evans blue dye to document the increased vascular permeability.Plasma exudation was assessed by blue coloring of foot pads 30 min afterthe antigen challenge (Oettgen et. al., 1994, Nature, 370:367-370).Vehicle-treated control mice showed a marked blue coloring of their footpads after antigen challenge but no significant blue coloring wasobserved in mice pretreated with the JAK3 inhibitor WHI-P131 (FIG. 14B).

Mast cell degranulation in histologic sections of ears was also assessedby examining their fluorescence intensity after staining withavidin-FITC. Avidin specifically binds to heparin, the majorproteoglycan in the granules of connective tissue mast cells (Malaviyaet. al., 1994, J. Clin. Invest., 93:1645-1653). The fluorescenceintensity of the stained mast cells is proportional to the amount ofheparin and therefore degranulation reduces the fluorescence intensity.Whereas the IgE/antigen challenge resulted in a marked reduction offluorescence intensity of avidin-FITC stained tissue mast cells ofcontrol mice consistent with degranulation-associated depletion ofheparin, no reduction in fluorescence intensity was observed for mastcells from WHI-P131 pretreated mice (FIG. 14C). Since the majorvasoactive mediator released from activated mast cells is histamine, andsystemic anaphylaxis in humans and rodents has been associated with asignificant increase in blood histamine levels (Oettgen et. al., 1994,Nature, 370:367-370; Miyajima et. al., 1997, J. Clin. Invest.,99:901-914), blood samples were obtained from mice 5 minutes after theantigen challenge to determine their plasma histamine levels (Malaviyaet. al., 1996a, J. Invest. Dermatol, 106:785-789). As expected, theantigen challenge resulted in marked elevation of plasma histaminelevels, but pretreatment with the JAK3 inhibitor WHI-P131 substantiallyreduced this histamine response (FIG. 14D). These results demonstratethat WHI-P131 is capable of preventing passive cutaneous and systemicanaphylaxis by blocking mast cell degranulation in vivo.

The efficacy of WHI-P131 was next tested in a model ofIgE/antigen-induced active systemic anaphylaxis. To this end, mice werefirst injected with BSA in an aluminum hydroxide gel to trigger aBSA-specific IgE response. Ten days later, these BSA-sensitized micewere rechallenged with this antigen to induce anaphylaxis. Eight of 15(53%) BSA-sensitized mice that were treated with WHI-P131 prior toantigen challenge survived without any signs of anaphylaxis, whereas 12of 12 control mice (100%) developed anaphylaxis within 45 minutes afterantigen challenge (P<0.0001 by log-rank test; FIG. 14E).

CONCLUSION

In summary, the studies detailed herein provide experimental evidencethat JAK3, a member of Janus family protein tyrosine kinases, plays apivotal role in IgE receptor-mediated mast cell responses. Furthermore,the data demonstrate that targeting JAK3 in mast cells with WHI-P131[4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline], a potent andspecific inhibitor of JAK3, abrogates mast cell degranulation andrelease of allergic mediators in vitro and, at nontoxic dose levels,prevents IgE receptor/FcεRI mediated anaphylactic reactions, includingfatal anaphylactic shock, in vivo.

Studies employing chimeric receptors and chimeric JAKs support thenotion that JAKs act primarily as conduits of signal transmission by anauthoritative cytokine receptor (Nelson et. al., 1996, Mol. Cell. Biol.,16:369-375). Recent studies suggest that individual JAKs may also havedistinct functions and promote unique signals by selectively recognizingspecific substrates (Endo et. al., 1997, Nature, 387:921-924; Witthuhnet. al., 1999, Lymphoma Leukemia, in press"). Janus kinase JAK3 has beenshown to play an important role for lymphocyte development, activation,and cytokine responsiveness (Ihle and Kerr, 1995, Trends Genet,11:69-74; Nosaka et. al., 1995, Science, 270:800-802). The present studyexpands knowledge of JAK3 functions and reveals that JAK3 has essentialand non-redundant functions for the full signaling capacity of the highaffinity IgE receptor on mast cells. This previously unknown function ofJAK3 provides a basis for new and effective treatment as well asprevention programs for mast cell mediated allergic reactions using JAK3inhibitors. Based on the data presented herein and previous reportsregarding the known function of Syk in IgE receptor/FcεRI mediatedresponses (Costello, et. al. 1996, Oncogene, 13:2595-2605; Oliver, et.al., 1994, J Biol Chem. 269:29697-29703), JAK3 and Syk may cooperate ininitiation of mast cell mediated hypersensitivity reactions.

Intriguingly, SYK is also activated by cytokines, such as IL-2, IL-3,and GMCSF, known to also activate the JAK/STAT pathway and is capable ofphosphorylating STATs (Matsuda and Hirano, 1994, Blood, 83:3457-3461).Similarly, JAK3 can modulate the function of the Syk substratePI3-kinase via phosphorylation of the insulin receptor substrateproteins IRS1 and IRS2 (Johnston et. al., 1995, J. Biol. Chem.,270:28527-30; Yamauchi et. al., 1998, J. Biol. Chem. 273:15719-15726).Another possible mechanism for crosstalk may be through JAK3interactions with Sam68, a Src substrate which associates with the SYKsubstrates PLCγ, PI3-kinase, Grb2, and Cbl as well (Yin et. al., 1995,J. Biol. Chem. 270:20497-502; Fusaki et. al., 1997, J. Biol. Chem.272:6214-6219; Deckert et. al., 1998, J. Biol. Chem., 273:8867-8874).

This investigation extends earlier studies on the role of PTK in mastcell responses (Blank et. al., 1989, Nature, 337:187-189; Oliver et.al., 1994, J Biol Chem. 269:29697-29703; Hamawy et. al., 1995, CellularSignalling 7:535-544; Scharenberg et. al., 1995, EMBO J., 14:3385-3395;Costello et. al., 1996, Oncogene, 13:2595-2605) and offers new evidencesupporting the therapeutic potential of PTK inhibitors in the treatmentof allergic disorders. Because of its in vivo potency and lack ofsystemic toxicity, WHI-P131 and structural analogs that fit the bindingpocket model, are not only the first JAK3-specific PTK inhibitors butalso the first PTK inhibitors to modulate mast cell functions both invitro and in vivo, and offer a new and effective treatment for mast cellmediated hypersensitivity reactions in clinical settings.

What is claimed is:
 1. A method for inhibiting JAK-3 tyrosine kinaseactivity comprising contacting JAK-3 tyrosine kinase with a compound offormula I: ##STR13## wherein X is HN, R₁₁ N, S, O, CH₂, or R₁₁ CH;R₁₁ ishydrogen, (C₁ -C₄)alkyl, or (C₁ -C₄)alkanoyl; R₁ -R₈ are eachindependently hydrogen, hydroxy, mercapto, amino, nitro, (C₁ -C₄)alkyl,(C₁ -C₄)alkoxy, (C₁ -C₄)alkylthio, or halo; wherein two adjacent groupsof R₁ -R₅ together with the phenyl ring to which they are attached mayoptionally form a fused ring, for example forming a naphthyl or atetrahydronaphthyl ring; and further wherein the ring formed by the twoadjacent groups of R₁ -R₅ may optionally be substituted by 1, 2, 3, or 4hydroxy, mercapto, amino, nitro, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, (C₁-C₄)alkylthio, or halo or R₉ and R₁₀ can independently be a prodrugderivative.
 2. The method of claim 1 wherein the JAK-3 inhibitor is acompound of formula II: ##STR14## where R3' is H or halo;R4' is H or OH;R5' is OH; R6' is H, OH, NO₂, NH₂, CH₂ OH; R9' and R10' are R₉ and R₁₀are each independently hydrogen, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, halo, or(C₁ -C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy or halo orR₉ and R₁₀ can independently be a producing derivative; or apharmaceutically acceptable salt thereof.
 3. The method of claim 1wherein the JAK-3 inhibitor is a compound of formula III: ##STR15##where: R5' is OH;R6' is halo; R9' and R10' are R₉ and R₁₀ are eachindependently hydrogen, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy or R₉ and R₁₀can independently be a producing derivative; or a pharmaceuticallyacceptable salt thereof.
 4. The method of claim 1 wherein the JAK-3inhibitor is a compound of formula IV: ##STR16## where: R5' is halo, OH,NH₂, NO₂, CH₂ OH;R9' and R10' are R₉ and R₁₀ are each independentlyhydrogen, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, halo, or (C₁ -C₄)alkanoyl; orR₉ and R₁₀ together are methylenedioxy or R₉ and R₁₀ can independentlybe a producing derivative; or a pharmaceutically acceptable saltthereof.
 5. The method of claim 1 wherein the JAK-3 inhibitor is acompound of formula V: ##STR17## where: R^(5') is halo;R^(7') is halo,OH, NH₂, NO₂, CH₂ OH; R^(9') and R^(10') are each independentlyhydrogen, (C₁ -C₄)alkyl, (C₁ -C₄)alkoxy, halo, or (C₁ -C₄)alkanoyl; orR^(9') and R^(10') together are methylenedioxy or R^(9') and R^(10') canindependently be a producing derivative; or a pharmaceuticallyacceptable salt thereof.
 6. The method of claim 1 wherein the JAK-3inhibitor comprises4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the JAK-3 inhibitor comprises4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.